Dissociation of the eukaryotic initiation factor-4E/4E-BP1 complex involves phosphorylation of 4E-BP1 by an mTOR-associated kinase. mTOR immunoprecipitates contain two 4E-BP1 protein kinase activities. One appears to be due to mTOR itself and results in the phosphorylation of 4E-BP1 on residues T(36) and T(45), as shown previously by others. The other is a kinase which can be separated from mTOR and which phosphorylates 4E-BP1 within a peptide(s) containing residues S(64) and T(69). This phosphorylation, which occurs predominantly on S(64), results in the dissociation of 4E-BP1 from eIF-4E.