Dibutyrylcytidine 3',5'-cyclic monophosphate (Bt2cCMP) stimulated neurite outgrowth in rat pheochromocytoma PC12 cells in a dose-dependent manner at the dose range from 0.5 mM to 6 mM. About 25% of the cells had neurites in response to 6 mM Bt2cCMP. In contrast with the effect of nerve growth factor (NGF) which increased the percentage of cells with neurites gradually during a 6-day culture period, the stimulating effect of Bt2cCMP reached a plateau 2 days after plating. Staurosporine inhibited the neurite outgrowth induced by NGF, but not that by Bt2cCMP. These results suggest that Bt2cCMP stimulates neuronal differentiation by a mechanism different from that by NGF.