The Russian Air Force bombed U.S.-trained rebels in southern Syria not once, but twice Thursday, and the second wave of attacks came after the U.S. military called Russia on an emergency hotline to demand that it stop, a defense official with knowledge of the attack tells Fox News.

"They came back and struck again," the official said about the Russians, visibly angry about the incident.

Defense Secretary Ash Carter raised some eyebrows at a Hack the Pentagon event at the Pentagon Friday by not condemning the strikes, saying he did not know if the Russians meant to hit these specific rebels or not.

"This was an attack on forces fighting ISIL and that is problematic,” Carter said, adding that faulty intelligence might be to blame.

"The Russians initially said that they were coming in to fight ISIL," Carter said. "If... it was their intention, that's the opposite of what they said they were going to do."

Carter said the communications hotline set up between the U.S. military and Russia "wasn’t professionally used."

For months, Pentagon officials have said publically the hotline was to be used to "de-conflict" airspace and not coordinate activities with the Russians, which raises more questions about the interactions between the U.S. government and Russia over the Syrian war.

Russian Su-34 "Fullbacks" conducted the airstrikes, according to the defense official who said it was most likely a flight of two, the typical attack profile for the Russian Air Force in Syria. Su-34s are modern Russian fighter-bombers comparable to a U.S. Air Force F-15.

The Russian Air Force "had never conducted strikes" in that portion of Syria before, the official said. Al-Tanf is nestled in an area where Syria, Jordan and Iraq come together. Some of the U.S.-backed Syrian rebels were part of the Pentagon's "train and equip" program, and others were CIA-trained, the official said.

The attack left some Syrian rebels dead and others wounded, the official said, but he did not have any specific numbers immediately available.

Moscow has long objected to the U.S. government covertly training anti-Assad forces and has killed hundreds of U.S.-backed fighters since Russia's air campaign began in Syria in late September. Fox News reported 150 such CIA-trained and equipped rebels had been killed by October 2015 according to U.S. officials close to the program.

The official said the wounded were receiving treatment in Jordan. He was not aware of any U.S. military involvement in the evacuation. He said the wounded most likely were taken by truck into Jordan.

There were no U.S. forces on the ground in the area where the Russians struck Thursday, the official said.

"This was a counter-ISIL" group, the official said describing the group as rebels trained to attack ISIS or the Islamic State.

The camp that was hit was a "staging area" for the U.S.-backed forces, the official said.

After the first Russian strike Thursday, the rebels called the Combined Air Operations Center located at Al Udeid Air Base in Qatar, where the U.S. military runs the air war against ISIS.

Immediately upon taking the call, senior U.S. military officials called their Russian counterparts on a hotline established between the U.S. and Russia following Russia's deployment of warplanes and helicopter gunships to Syria late last year.

The line is tested daily and the Pentagon routinely conducts video teleconferences with their Russian counterparts in Moscow to discuss the agreement.

Meanwhile, President Vladimir Putin's spokesman Dmitry Peskov said Friday that an attempt to topple Assad's government "wouldn't help a successful fight against terrorism and could plunge the region into total chaos."

Peskov made the statement while asked to comment about an internal document in which dozens of State Department employees called for military action against Assad's forces.

President Barack Obama called for regime change in Syria early on in the five-year conflict, but so far has only authorized strikes against the Islamic State group and other U.S.-designated terror groups in Syria.

The Associated Press contributed to this report.

Lucas Tomlinson is the Pentagon and State Department producer for Fox News Channel. You can follow him on Twitter: @LucasFoxNews