UCN-01 is a derivative of staurosporine, initially developed as a potentially selective inhibitor of the Ca²⁺- and phospholipid-dependent protein kinase C, but with the capacity to inhibit a number of tyrosine and serine/threonine kinases. UCN-01 inhibits the growth of 5 breast carcinoma cell lines with a 50% inhibitory concentration range of 30–100 nm during 6 days of continuous exposure. In MCF-7, MDA-MB453, and SK-BR-3 cells, UCN-01 is 5-fold more potent in growth inhibition than its diastereomer UCN-02, but the 2 compounds are equipotent in the inhibition of MDA-MB468 and H85787 cell growth. A differential sensitivity to a 24-h period of exposure to UCN-01 followed by drug removal and growth for 5 subsequent days was observed. The rank order for persistent inhibition of cells by UCN-01 was MCF-7, MDA-MB453 >> SK-BR-3 > H85787 > MDA-MB468. MCF-7 and MDA-MB453 cells did not resume proliferation within the 5 days after brief exposure to UCN-01. In contrast, MDA-MB468 and H85787 cells showed no net growth inhibition after a 24-h pulse of UCN-01, followed by 5 more days of growth in drug-free medium. In MDA-MB468 cells, 150 nm UCN-01 retards but does not prevent cell cycle progression through S phase, but the cells are clearly blocked from exit of G₁ and entry into S. Progression through S phase is completely inhibited by 600 nm UCN-01. The development of a G₁ to S block by UCN-01 in MDA-MB468 cells occurs in conjunction with inhibition of [₃₂P]orthophosphate labeling and decreased phosphotyrosine mass of discrete cellular phosphoproteins.