Multidrug resistance (MDR) is frequently associated with overexpression of a 170-kDa P-glycoprotein (Pgp). Data suggest altered protein kinase C (PKC) activity in cells expressing the multidrug-resistant phenotype. The staurosporine derivative CGP 4 I25 I, an experimental anticancer drug, has been shown to exert selectivity for inhibition of protein kinase C activity and to exhibit antitumor activity in vitro and in vivo. Here we show that CGP 4 I25 I is also able to reverse MDR. After treatment of the multidrug-resistant human lymphoblastoid cell line CCRF-VCRlOOO with 500 nM Adriamycin, cell proliferation was re- duced to 8 I 'Yo of untreated controls. A combination of 500 nM Adriamycin with a non-toxic concentration of 150 nM CGP 41251 (ICso for inhibition of cell proliferation 420 nM CGP 4 I25 I) inhibits cell proliferation of CCRF-VCRl000 cells to 29% of untreated controls. In sensitive CCRF-CEM cells no enhancement of Adriamycin-induced cytotoxicity was observed upon addition of 150 nM CGP 41251. Strong synergism of the inhibition of cell proliferation was also observed after concomitant treatment of KB-85 I I cells with CGP 4 I25 I and Vinblastine or Adriamycin. Drug-sensitive KB-31 cells could not be further sensitized to Adriamycin or Vinblastine with CGP 4 I25 I doses above 100 nM. Pretreatment with 50-1000 nM CGP 41251 for 30 min led to a dose-dependent increase in the intracellular accumulation of rhodamine 123, a substrate of P-glycoprotein. Treatment of multidrug-resistant CCRF-VCR1000 cells with CGP 41251 for 10 min was sufficient to inhibit the efflux of rhodamine 123. Preincubation with CGP 41251 for 12 or 24 hr did not alter multidrug resistance gene (mdrI)-mRNA levels. CGP 41251, a drug with antitumor efficacy in experimental systems, might offer an attractive combination partner for the treatment of tumors expressing the MDR phenotype.