Eric Brunner, recently released by MLS's New York Red Bulls, and Sammy Ochoa of Mexican club UAG's Second Division team, both scored in their U-23 debuts.
Peter Nowak's Americans were down by two goals for most of the match, cutting the lead to one on two occasions, but was ultimately unable to come back from the early deficit.
The only player in the starting lineup against Turkey who took part of the USA's successful run through CONCACAF Olympic Qualifying in March was goalkeeper Dominic Cervi.
Captain Benny Feilhaber left the game in the 67th minute with a knee injury and will be evaluated on Thursday in England after he joins the men's national team for next Wednesday's England-USA game.
"I thought in the first half we came out a little nervous, which is a natural thing," said Nowak. "We were playing for the first time together. We came here to this tournament to gain experience and to have a look at different players. The group trained together for only five days, so for their first time it wasn't a bad effort."
The USA will face Ivory Coast on Friday (kickoff: 12:30 p.m. ET), while Italy and Turkey will meet in the second match of the double-header in La Seyne. Both games will be broadcast live on Fox Soccer Channel.
May 21 in Toulon
Turkey 3 USA 2. Goals: Eren 18, Nuri 35, Ilhan 59; Brunner 58, Ochoa 72.
USA -- Cervi, Wagner (Beltran, 46), Brunner, Valentin, Leathers, Ashe, Feilhaber (Shea, 67), Dalby, Nguyen (Hill, 46); Ochoa, Ferrari.
Turkey -- Ufuk, Durmus, Eren, Orhan, Erkin, Mehmet (Zafer, 57), Ozer, Nuri, Serdar (13-Bilal, 68), Adem, Ilhan.
Referee: Kenji Ogiya (Japan).