Antitumor activity of UCN-01 (7-hydroxy staurosporine), a selective inhibitor of Ca2+- and phospholipid-dependent protein kinase C, was examined in comparison with staurosporine, a nonselective inhibitor of protein kinases, on human and murine tumor cell lines which have some aberrations in cellular signal transduction. UCN-01 inhibited the growth of five tumor cell lines about 9 to 90 times less potently than staurosporine in vitro. UCN-01 showed an in vivo antitumor effect against three human tumor xenografts [epidermoid carcinoma A431 (c-erbB-1 overexpression), fibrosarcoma HT1080 (N-ras activation), and acute myeloid leukemia HL-60 (N-ras activation)], giving a minimum treated/control ratio of 0.40 (P less than 0.01), 0.17 (P less than 0.01), and 0.61 (P less than 0.05), respectively. UCN-01 also exhibited significant antitumor activity against two murine tumor models (fibrosarcoma, K-BALB and M-MSV-BALB), which activated the v-ras and v-mos oncogenes, showing a minimum treated/control ratio of 0.27 (P less than 0.01) and 0.21 (P less than 0.01). Staurosporine did not show significant antitumor activity against any of these five tumors. UCN-01 inhibited the down-modulation of epidermal growth factor receptor caused by phorbol 12-myristate 13-acetate in A431 cells at a near 50% inhibitory concentration for cell growth. These results imply that UCN-01 is a promising antitumor agent which has a novel mechanism(s) of action.