The macrolide rapamycin blocks cell cycle progression in yeast and various animal cells by an unknown mechanism. We demonstrate that rapamycln blocks the phosphorylatlon and activation of the 70 kd S6 protein klnases (~~ 708”) in a variety of animal cells. The structurally related drug FK506 had no effect on~~ 70~ activation but at high concentrations reversed the rapamycln-induced block, confirming the requirement for the rapamycln and FK506 receptor, FKBP. Rapamycin also interfered with signaling by these S6 klnases, blocking serum-stimulated S6 phosphorylatlon and delaying entry of Swiss 3T3 cells into S phase. Neither rapamycln nor FK506 blocked activation of a distinct family of S6 kinases (RSKs) or the MAP kinases. These studies identify a rapamycin-sensitive signaling pathway, argue for a ubiquitous role for FKBPs in signal transductfon, indicate that FK506-FKBP-calcineurln complexes do not interfere with~~ 70~ signaling, and show that in flbroblasts pp7p, not RSK, is the physiological S6 kinase.