UCN-01 (7-hydroxyl-staurosporine) was originally isolated as a Ca²⁺- and phospholipid-dependent protein kinase C selective inhibitor and now is being developed as an anticancer agent. Results from our and other laboratories have suggested that UCN-01 induces preferential G₁-phase accumulation in several human tumor cell lines tested. To elucidate this mechanism, we examined the effects of UCN-01 on several cell cycle-regulatory proteins critical for G₁-S-phase transition in p53-mutated human epidermoid carcinoma A431 cells. After 24 h exposure at around 50% growth-inhibitory concentrations (IC₅₀s), 260 and 520 nm, UCN-01 induced the accumulation of pRb (the dephosphorylated retinoblastoma protein form). The protein expression of cyclin A but not cyclin E was markedly reduced and that of cyclin D1 was partially reduced under the same condition. UCN-01 also showed the concentration-dependent inhibitions of the activity of cyclin-dependent kinase 2 (CDK2) using histone H1 and pRb as substrates in vitro (IC₅₀, 530 and 640 nm, respectively). In addition, CDK2 activities of the cells pretreated with UCN-01 for 24 h at 260 and 520 nm were markedly inhibited, giving IC₅₀s of far less than 260 nm. When the same cell lysates were analyzed by Western blotting for CDK2, the lower band (e.g., active and phosphorylated CDK2) was remarkably reduced, in accordance with the reduced activity. Furthermore, UCN-01 induced the expression of the CDK inhibitor p21 protein and its complex formation with CDK2 after 24 h exposure at 260 and 520 nm, whereas the expression level was very low or undetectable in untreated or DNA-damaged cells. The increase of p21 mRNA levels was also induced under the same condition. UCN-01 further increased luciferase activities in A431 cells transiently transfected with p21 promoter-luciferase reporter plasmid after 24 h exposure at 260 and 520 nm. UCN-01 also increased the expression of the CDK inhibitor p27 protein after 24 h exposure at 260 and 520 nm. These results suggest that G₁-phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitors p21 and p27.