The mTORC1 complex (mTOR-raptor) is modulated by mitogen-activated protein (p44/42 MAP) kinases (p44/42) through phosphorylation and inactivation of the tuberous sclerosis complex. However, a role for mTORC1 signaling in modulating activation of p44/42 has not been reported. We show that in two cancer cell lines regulation of the p44/42 MAP kinases is mTORC1-dependent. In Rh1 cells rapamycin inhibited insulin-like growth factor-I (IGF-I)–stimulated phosphorylation of Thr202 but not Tyr204 and suppressed activation of p44/42 kinase activity. Down regulation of raptor, which inhibits mTORC1 signaling, had a similar effect to rapamycin in blocking IGF-1-stimulated Tyr204 phosphorylation. Rapamycin did not block maximal phosphorylation of Tyr204, but retarded the rate of dephosphorylation of Tyr204 following IGF-1 stimulation. IGF-1 stimulation of MEK1 phosphorylation (Ser217/221) was not inhibited by rapamycin. Higher concentrations of rapamycin (= 100 ng/ml) were required to inhibit EGF-induced phosphorylation of p44/42 (Thr202). Rapamycin-induced inhibition of p44/42 (Thr202) phosphorylation by IGF-I was reversed by low concentrations of okadaic acid, suggesting involvement of protein phosphatase 2A (PP2A). Both IGF-I and EGF caused dissociation of PP2A catalytic subunit (PP2Ac) from p42. Whereas low concentrations of rapamycin (1 ng/ml) inhibited dissociation of PP2Ac after IGF-I stimulation, it required higher concentrations (=100 ng/ml) to block EGF-induced dissociation, consistent with the ability for rapamycin to attenuate growth factor-induced activation of p44/42. The effect of rapamycin on IGF-I or insulin activation of p44/42 was recapitulated by amino acid deprivation. Rapamycin effects altering the kinetics of p44/42 phosphorylation were completely abrogated in Rh1mTORrr cells that express a rapamycin resistant mTOR, whereas the effects of amino acid deprivation were similar in Rh1 and Rh1mTORrr cells. These results indicate complex regulation of p44/42 by phosphatases downstream of mTORC1. This suggests a model in which mTORC1 modulates the phosphorylation of Thr202 on p44/42 MAP kinases through direct or indirect regulation of PP2Ac.