Protein kinases play an important role in the response of mammalian cells to ionizing radiation. In this study, we examined the effect of staurosporine, a potent inhibitor of protein kinases, on cell survival after X-irradiation, using the normal rat fibroblast line 3Y1. Treatment with 30 ng/ml staurosporine 1 h before irradiation resulted in the increase in survival of 3Y1 cells. This phenomenon was drug dose-dependent and maximal reduction of radiation-induced cell killing occurred when more than, or equal to, 30 ng/ml staurosporine was added. In contrast, treatment with 30 ng/ml staurosporine after irradiation did not increase cell survival. There was no change in cell cycle distribution by treatment for 1 h with 30 ng/ml staurosporine. These data suggest that the inhibition of protein kinase activities by staurosporine influences the radiosensitivity of 3Y1 cells.