Inactivation of the retinoblastoma (Rb) tumor suppressor protein is essential for the G1/S transition during mammalian cell cycle progression. Although Rb is inactivated by phosphorylation by cyclins D and E and their associated kinases during cell cycle progression, we find that Rb is inactivated upon apoptotic stimulation by Fas through the mediation of p38 kinase, independent of cyclins and cyclin-dependent kinases (cdks). Inactivation by p38 kinase coincided with increased phosphorylation of Rb leading to dissociation of E2F and increased transcriptional activity; such p38-mediated changes in Rb function occurred only during Fas stimulation but not mitogenic progression. p38 kinase targets Rb preferentially and had minimal effects on p107 and had no effect on p130 function. We also find that phosphorylation site mutants of Rb (PSM7LP and PSM9-Rb) that cannot be inactivated by cdks can be targeted by Fas and p38 kinase, suggesting that Rb inactivation by these kinases is biochemically and functionally distinct. It appears that Rb inactivation is achieved by different kinase cascades in response to mitogenic and apoptotic signals.