Control of the coastal oil town of Brega has swung back and forth
They say it was launched from near the coastal city of Sirte
and aimed at Brega - currently held by rebels - but landed harmlessly in
the desert.
Pro-Gaddafi forces are believed to have more than 200 Scuds in their arsenal.
The move comes as the rebels continue a push towards the capital, Tripoli.
On Monday they advanced into two strategically important towns controlling access to the capital, from the west and the south.
Fighting was reported in both Zawiya, 50km (30 miles) west of Tripoli, and Gharyan, 80km (50 miles) to the south.
Medics outside Zawiya told Reuters news agency that sniper
and mortar fire from government troops had left three civilians dead.
'Days numbered'
Col Gaddafi's forces are still thought to control the Zawiya
oil refinery - the only one in western Libya - where they get most of
their fuel.
Earlier, the rebels said they had seized Gharyan - a town in
the Nafusa Mountains that straddles the road connecting Tripoli with
Sabha, Sorman - west along the road from Zawiya to Tunisia - and Tiji,
although this has not been confirmed by residents.
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Libya conflict: Rebels fight for key towns near Tripoli
If the towns fell to the rebels, they would have Tripoli surrounded by land, with Nato blocking sea access.
The BBC's Matthew Price in Tripoli says how much territory the rebels hold and for how long they can hold it is unclear.
But both rebels and the US said Col Gaddafi was coming under increasing pressure.
Rebel spokesman Ahmed Bani said the use of a Scud was a sign
of desperation by the Libyan leader. "This man will use any weapon
necessary to keep his regime going and resume his rule, even for few
hours," he said.
"It's becoming increasingly clear that Gaddafi's days are numbered," said White House spokesman Jay Carney.
Benjamin Barber, a Libya expert and fellow at the think tank Demos, said such claims were "wishful thinking".
"I don't think there's any particular reason for hope unless
they're willing to do a negotiated settlement, but neither the
opposition council, nor Nato, nor the United States seem very interested
in that right now," he told the BBC World Service.
"There are a great many tribes and groups in and around Tripoli whose future depends on the survival of Gaddafi," he added.
"I think this is more wishful thinking to cover the fact that
the West, Europe, Nato, and the United States have been unwilling to
make the sort of deals that were on the table that would have allowed
Gaddafi to step away from power but stay in Libya."
In an audio message broadcast on Libyan state TV on Monday,
Col Gaddafi was defiant. "The end of the coloniser is close and the end
of the rats is close," he said.
He appealed to his supporters to "prepare for the battle to liberate" Libya.
Tunisia talks?
The Scud launch was detected by US forces, who say the missile
was fired on Sunday morning and landed in the desert about 80km (50
miles) outside the strategic port of Brega.
The two sides have battled over Brega, with control swinging back and forth.
Scud missiles have a range of up to about 800km (500 miles) and can carry a warhead of up to about one tonne.
Earlier in the conflict the Nato-led coalition targeted
rocket launchers and Scud missile containers near Sirte, saying the
weapons could be used against civilians or on areas beyond Col Gaddafi's
control.
Rebels denied that their National Transitional Council (NTC) has held talks with representatives of Col Gaddafi in Tunisia.
"There are no negotiations or talks between the Gaddafi
regime and the NTC in Tunisia or anywhere else," Abdel Hafiz Ghoga, NTC
vice-chairman, told AFP news agency.
A UN special envoy, Abdul Ilah al-Khatib, whose role is to
negotiate a ceasefire, has arrived in Tunis, and sources in the Tunisian
security services said the two sides had met at a hotel on the
Mediterranean resort island of Djerba on Sunday.
A UN spokesman said he had "no concrete information" on any such talks.
Meanwhile, the Netherlands on Monday unfroze $143m (100m
euros; £87m) in seized Libyan assets and gave the funds to the World
Health Organization.
A spokesman for the Dutch government said the funds would be
used to provide medicines and surgical equipment in areas held by
rebels.
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