The mammalian target of rapamycin, mTOR, has been shown to be an upstream regulator of translational effectors. In the present study, in order to detect potential molecules involved in the mTOR signaling, an in vitro phosphorylation assay using mTOR immunoprecipitates from HEK293 cells was carried out. In addition to the autophosphorylation of mTOR, 32P incorporation into 80-kDa (pp80) and 175-kDa (pp175) bands was observed in mTOR immunoprecipitates. The protein kinase activity toward the recombinant eIF-4E binding protein 1 (4E-BP1) was also detected as previously described. When mTOR immunoprecipitates from HEK293 cells were prepared in the presence of a detergent, Nonidet P-40, the 4E-BP1 kinase activity and 32P incorporation into pp175 dramatically diminished, while the phosphorylation of mTOR and 32P incorporation into pp80 did not change. These results raised a possibility that mTOR may associate with protein cofactors, some of which may be involved in the regulation of kinase activities associated with mTOR.