Some, but not all, studies have suggested that high-fat diets promote colon carcinogenesis, possibly by stimulating the proliferative activity of colonic epithelium. Both the increase in colonic excretion of bile salts and of fatty acids that occur with an increase in fat ingestion have been implicated as stimuli of epithelial proliferative activity. In this study, we examined the role of activation of protein kinase C in fatty acid-induced stimulation of colonic epithelial proliferation in the rat. Intracolonic instillation of arachidonate, linoleate, or oleate at concentrations that did not induce surface cell injury or loss increased colonic mucosal ornithine decarboxylase activity and stimulated incorporation of [3H]thymidine into mucosal deoxyribonucleic acid. The saturated fatty acid palmitate was without effect. Arachidonate, linoleate, and oleate each induced the translocation of protein kinase C activity from the soluble fraction to the membrane fraction of colonic mucosa, an index of enzyme activation. The translocation of protein kinase C induced by unsaturated fatty acids occurred both in vivo after intracolonic instillation of these agents and in vitro upon incubation of isolated colonic crypt epithelium with fatty acids. The effects of the unsaturated fatty acids on both enzyme translocation and colonic epithelial proliferative activity were suppressed by 1-(5-isoquinolinyl)-2-methylpiperazine, an inhibitor of protein kinase C activity. Unsaturated fatty acids directly stimulated soluble colonic mucosal protein kinase C activity when added to the enzyme assay mixture. This action was blocked by 1-(5-isoquinolinyl)-2-methylpiperazine. However, unsaturated fatty acids also increased the breakdown of polyphosphoinositides when added to isolated colonic epithelium. The increase in polyphosphoinositide breakdown resulted in release of diacylglycerol, an endogenous activator of protein kinase C. Thus, unsaturated fatty acids may activate protein kinase C of colonic epithelium through either a direct intracellular effect or through an action on the cell membrane. The results support a role for protein kinase C in the stimulation of colonic epithelial proliferation by unsaturated fatty acids.