Activation of p70s6k in cells stimulated with serum correlates with the phosphorylation of seven sites. Pretreatment of Swiss 3T3 cells with the immunosuppressant rapamycin blocks phosphorylation of four of these sites (Thr229, Thr389, Ser404, and Ser411), whereas phosphorylation proceeds in the remaining three sites (Ser418, Thr421, and Ser424). If rapamycin is added postserum stimulation, the pattern of phosphorylation is qualitatively similar except that Ser411 is still highly phosphorylated. The inhibitory effect of rapamycin on serum-induced p70s6k activation and the phosphorylation of Thr229, Thr389, Ser404, and Ser411 is rescued by FK506, providing further evidence that the inhibitory effect is exerted through a complex of rapamycinFKBP12. Wortmannin treatment pre- or post-serum stimulation inhibits phosphorylation of the same set of sites as rapamycin, supporting the argument that both agents act on the same pathway. Likewise, methylxanthine phosphodiesterase inhibitors block p70sek activation and phosphorylation of the same set of sites as wortmannin and rapamycin. However, other agents that raise intracellular cAMP levels have no inhibitory effect, leading to the hypothesis that the inhibitory actions of methylxanthines on p70s6k activity are not through activating protein kinase A but through inhibition of an upstream kinase. Together the results indicate that there are two kinase signaling pathways that must converge to activate p70sek and that only one of these pathways is sensitive to rapamycin, wortmannin, and methylxanthine inhibition.