Rapamycin (RAP) has recently been shown to inhibit the phosphorylation and activity of p70 S6 kinase (p70s6k). In interleukin (IL)-2-induced cell activation of the human IL-2-dependent T-cell line, Kit225, RAP inhibited p70s6k phosphorylation and activation, but not the activation of MAP kinase, p90 S6 kinase (p90rsk), early tyrosine kinases, or the transcription of the c-fos and c-myc genes. Cell cycle progression induced by IL-2 was arrested by RAP prior to p110Rb phosphorylation and the major increase in total RNA synthesis, both of which were initiated around 6 h after addition of IL-2 and 9 h before the beginning of DNA synthesis. Interestingly, RAP could not inhibit DNA synthesis if addition of the drug was delayed for 6 h after addition of IL-2, despite the fact that even at this time, RAP rapidly induced the accumulation of the dephosphorylated form of p70s6k and that p70s6k was inactivated within 1 h of RAP addition. Furthermore, when RAP was added to continuously growing Kit225 cells, cell proliferation was maintained for at least two additional cell cycles, in the absence of apparent p70s6k activity. These results indicate that 1) among the earliest detectable signals after IL-2 treatment, RAP selectively inhibits p70s6k activation, 2) RAP inactivates p70s6k regardless of the stage of the cell cycle in which the drug is added, 3) RAP blocks resting T-cells from entering the cell cycle, but does not directly arrest cell cycle progression once cells have entered the cycle, and 4) inactivation of p70s6k does not cause immediate arrest of cell cycle progression once cells have entered the cycle.