Senescent human diploid fibroblasts are unable to enter S phase in response to mitogenic stimulation. One of the key deficiencies in mitogen-stimulated senescent cells is their failure to phosphorylate the retinoblastoma protein, which acts as an inhibitor of entry into S phase in its unphosphorylated form. Recent data suggest that cyclin-dependent kinases (Cdks) regulated by G1 cyclins (D type and E) are responsible for the primary phosphorylation of the retinoblastoma protein prior to the G1/S boundary. Surprisingly, we found 10- to 15-fold higher constitutive amounts of both cyclin E and cyclin D1 in senescent cells compared to quiescent early-passage cells. Nevertheless, cyclin E-associated kinase activity in senescent cells was very low and did not increase significantly upon mitogenic stimulation even though cyclin E-Cdk2 complexes were abundant. In contrast to early-passage cells in late G1 phase, senescent cells contained mainly underphosphorylated cyclin E and proportionally more unphosphorylated and inactive Cdk2, perhaps accounting for the low kinase activity. We also show that a majority of the Cdk2 in senescent cells, but not in early-passage cells, was complexed with cyclin D1. Cyclin D1-Cdk2 complexes, severalfold enriched in senescent cells, contained exclusively unphosphorylated Cdk2. Amounts of cyclin A, which ordinarily accumulates in S and G2 phases, were extremely low in stimulated senescent cells. We suggest that the failure to activate cyclin E-Cdk2 kinase activity in senescent cells may account for the inability of these cells to phosphorylate the retinoblastoma protein in late G1 phase, which in turn may block the expression of late G1 genes such as cyclin A that are required for entry into S phase.