A critical step in S6 kinase 1 (S6K1) activation is Thr229 phosphorylation in the activation loop by the phosphoinositide-dependent protein kinase (PDK1). Thr229 phosphorylation requires prior phosphorylation of the Ser/Thr-Pro sites in the autoinhibitory domain and Thr389 in the linker domain, consistent with PDK1 more effectively catalyzing Thr229 phosphorylation in a variant harboring acidic residues in these positions (S6K1-E389D3E). S6K1-E389D3E has high basal activity and exhibits partial resistance to rapamycin and wortmannin, and its activity can be further augmented by mitogens, effects presumably mediated by Thr229 phosphorylation. However, PDK1-induced Thr229 phosphorylation is reported to be constitutive rather than phosphatidylinositide 3,4,5-trisphosphate-dependent, suggesting that S6K1-E389D3E activity is mediated through a distinct site. Here we use phosphospecific antibodies to show that Thr229 is fully phosphorylated in S6K1-E389D3E in the absence of mitogens and that regulation of S6K1-E389D3E activity by mitogens, rapamycin, or wortmannin parallels Ser371 phosphorylation. Consistent with this observation, a dominant interfering allele of the mammalian target of rapamycin, mTOR, inhibits mitogen-induced Ser371 phosphorylation and activation of S6K1-E389D3E, whereas wild type mTOR stimulates both responses. Moreover, in vitro mTOR directly phosphorylates Ser371, and this event modulates Thr389 phosphorylation by mTOR, compatible with earlier in vivo findings.