The senescence checkpoint constrains the proliferative potential of normal cells in culture to a finite number of cell doublings. In this study, we investigated the mechanism of cyclin dependent kinase (cdk)inhibition in senescent human prostatic epithelial cells(HPECs). Progression of HPECs from early passage to senescence was accompanied by a gradual loss of cells in S phase and an accumulation of cells containing 2N DNA. Furthermore, G₁-S phase-associated kinase activities progressively diminished with increasing cell passage. In senescent HPECs, cdk4 and cyclin E1- and A-associated kinases were catalytically inactive. In contrast to observations in senescent fibroblasts, levels of the kinase inhibitor protein (KIP) inhibitor p21^CIP1 diminished over the proliferative life span of HPECs. p27^KIP1 levels fell as cells approached senescence, and the association of both p21^CIP1 and p27^KIP1 with cdk4/6 complexes was decreased. However, the level of cyclin E1-associated KIP molecules was unaltered as cells progressed into senescence. Progression to senescence was accompanied by a progressive increase in both the level of p16^INK4A and in its association with cdk4 and cdk6. As HPECs approached senescence, cdk4- and cdk6-bound p16^INK4Ashowed a shift to a slower mobility due to a change in its phosphorylation profile. As p16^INK4A increased in cdk4 and cdk6 complexes, there was a loss of cyclin D1 binding. The altered phosphorylation of p16^INK4A in senescent prostatic epithelial cells may facilitate its association with cdk4 and cdk6 and play a role in the inactivation of these kinases.