The regulation of translation initiation is crucial for controlling cell growth and proliferation. The eIF4F complex, which recruits the 40S ribosomal subunit to the 5' end of mRNA, is inhibited by eIF4E-binding proteins (4E-BPs). The binding of 4E-BPs to eIF4E is regulated by phosphorylation: hypophosphorylated 4E-BP isoforms strongly interact with eIF4E, while hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells but hyperphosphorylated following exposure to various extracellular stimuli. The PI3-kinase/Akt pathway and the FRAP/mTOR kinase signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro, but it is unclear if it is responsible for all phosphorylation sites and whether phosphorylation at these sites leads to the release of 4E-BP1 from eIF4E. This study used recombinant FRAP/mTOR and a FRAP/mTOR immunoprecipitate in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping identified two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo, containing phosphorylated Thr-37 and Thr-46. These sites are efficiently phosphorylated by FRAP/mTOR in the presence of eIF4E, but phosphorylation does not disrupt the eIF4E/4E-BP1 complex. Mutational analysis showed that phosphorylation of Thr-37 and Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. These results suggest that phosphorylation of Thr-37 and Thr-46 by FRAP/mTOR is a priming event for subsequent phosphorylation of the carboxy-terminal sites. A two-step model is proposed, where FRAP/mTOR first phosphorylates Thr-37 and Thr-46, and then an unidentified serum-sensitive kinase phosphorylates the carboxy-terminal sites, leading to the release of 4E-BP1 from eIF4E and increased translation.The regulation of translation initiation is crucial for controlling cell growth and proliferation. The eIF4F complex, which recruits the 40S ribosomal subunit to the 5' end of mRNA, is inhibited by eIF4E-binding proteins (4E-BPs). The binding of 4E-BPs to eIF4E is regulated by phosphorylation: hypophosphorylated 4E-BP isoforms strongly interact with eIF4E, while hyperphosphorylated isoforms do not. 4E-BP1 is hypophosphorylated in quiescent cells but hyperphosphorylated following exposure to various extracellular stimuli. The PI3-kinase/Akt pathway and the FRAP/mTOR kinase signal to 4E-BP1. FRAP/mTOR has been reported to phosphorylate 4E-BP1 directly in vitro, but it is unclear if it is responsible for all phosphorylation sites and whether phosphorylation at these sites leads to the release of 4E-BP1 from eIF4E. This study used recombinant FRAP/mTOR and a FRAP/mTOR immunoprecipitate in in vitro kinase assays to phosphorylate 4E-BP1. Phosphopeptide mapping identified two 4E-BP1 phosphopeptides that comigrated with phosphopeptides produced in vivo, containing phosphorylated Thr-37 and Thr-46. These sites are efficiently phosphorylated by FRAP/mTOR in the presence of eIF4E, but phosphorylation does not disrupt the eIF4E/4E-BP1 complex. Mutational analysis showed that phosphorylation of Thr-37 and Thr-46 is required for subsequent phosphorylation of several carboxy-terminal serum-sensitive sites. These results suggest that phosphorylation of Thr-37 and Thr-46 by FRAP/mTOR is a priming event for subsequent phosphorylation of the carboxy-terminal sites. A two-step model is proposed, where FRAP/mTOR first phosphorylates Thr-37 and Thr-46, and then an unidentified serum-sensitive kinase phosphorylates the carboxy-terminal sites, leading to the release of 4E-BP1 from eIF4E and increased translation.