UCN-01 (7-hydroxyl-staurosporine), which was initially developed as a selective protein kinase C inhibitor, has an anti-tumor effect on several human cancer cell lines in vivo. In this study, we examined whether this compound has an inhibitory effect on cell cyclin-dependent kinases (cdks) in vitro and in vivo using A549 human lung adenocarcinoma cell line. UCN-01 inhibited the retinoblastoma susceptibility gene product (pRB) kinase activity of three types of cdks (cdk 2, 4 and 6) with 50% inhibitory concentration values of 42, 32, and 58 nM, respectively, in vitro. Moreover, the amount of phosphorylated pRB was reduced by UCN-01 at a concentration of 100 nM in the living cells. Flow cytometric analysis showed that UCN-01 inhibited cell cycle progression at G1 to S transition in A549 cells at the concentration of 100 nM. These results suggest that inhibition of pRB phosphorylation by UCN-01 might lead to inhibition of the cell cycle and thereby contribute to antitumor activity of this compound.