The mammalian target of rapamycin, mTOR, is a Ser/Thr kinase that promotes cell growth and proliferation by activating ribosomal protein S6 kinase 1 (S6K1). We previously identified a conserved TOR signaling (TOS) motif in the N terminus of S6K1 that is required for its mTOR-dependent activation. Furthermore, our data suggested that the TOS motif suppresses an inhibitory function associated with the C terminus of S6K1. Here, we have characterized the mTOR-regulated inhibitory region within the C terminus. We have identified a conserved C-terminal "RSPRR" sequence that is responsible for an mTOR-dependent suppression of S6K1 activation. Deletion or mutations within this RSPRR motif partially rescue the kinase activity of the S6K1 TOS motif mutant (S6K1-F5A), and this rescued activity is rapamycin resistant. Furthermore, we have shown that the RSPRR motif significantly suppresses S6K1 phosphorylation at two phosphorylation sites (Thr-389 and Thr-229) that are crucial for S6K1 activation. Importantly, introducing both the Thr-389 phosphomimetic and RSPRR motif mutations into the catalytically inactive S6K1 mutant S6K1-F5A completely rescues its activity and renders it fully rapamycin resistant. These data show that the N-terminal TOS motif suppresses an inhibitory function mediated by the C-terminal RSPRR motif. We propose that the RSPRR motif interacts with a negative regulator of S6K1 that is normally suppressed by mTOR.