As the colonic epithelium is physiologically exposed to butyrate and to activators of protein kinase C, we examined the effect of the protein kinase C signalling pathway on butyrate-induced expression of markers of differentiation. Activators and inhibitors of protein kinase C were used in combination with butyrate and effects on the expression of markers of differentiation examined in colon cancer cell lines. When the protein kinase C activator phorbol myristate acetate (100 nM) was added for 24 h prior to the addition of 2 mM butyrate, there was a synergistic increase in alkaline phosphatase activity (154 ± 11% above that for butyrate alone, P = 0.003) in a concentration- and time-dependent manner. Butyrate-induced expression of carcinoembryonic antigen and interleukin-8, dome formation and cell turnover were also markedly augmented by pre-treatment with phorbol myristate acetate. A similar effect was observed with propionate or acetate (but not other differentiating agents), when phorbol myristate acetate and butyrate were added concurrently, or when other protein kinase C activators were used. Pharmacological inhibition of protein kinase C activity did not alter butyrate-induced alkaline phosphatase activity, but abrogated the augmentation induced by phorbol myristate acetate. We conclude that protein kinase C does not mediate the differentiating effects of butyrate on colon cancer cells, but its activation regulates butyrate-induced cellular differentiation.