We have used short-term (8 h) cultures of week-old rat cerebellar granule cells to examine the effects on neuritogenesis of activation and down-regulation of protein kinase C by phorbol esters. We have previously demonstrated that endogenously released glutamate promoted neurite outgrowth in the same system acting via N-methyl-D-aspartate receptors. Low levels (0.1-1 nM) of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) evoked increases in the number of granule cells which extended neurites; higher levels (10-250 nM) which caused a down-regulation of total protein kinase C, inhibited outgrowth in a dose-dependent manner. N-Methyl-D-aspartate by itself also stimulated process outgrowth but could not reverse the inhibition evoked by either TPA or the protein kinase C inhibitor sphingosine. Stimulation of protein kinase C with 0.1 nM TPA resulted in a general increase in the incorporation of 32P-labelled inorganic orthophosphate into granule cell polypeptides. The results indicate that the activation of protein kinase C is involved in neuritogenesis in granule cells and are consistent with the idea that N-methyl-D-aspartate receptor activation may exert its effect on neuritogenesis through protein kinase C.