Phorbol 12,13-dibutyrate, an activator of protein kinase C, stimulates electrogenic anion secretion in chicken ileum. This effect can be inhibited by the cyclooxygenase inhibitor piroxicam, suggesting arachidonic acid metabolites as mediators. Phorbol 12,13-dibutyrate stimulates increases in prostaglandin E2 release and mucosal 3',5'-cyclic adenosine monophosphate content in intact mucosa. Stimulation of prostaglandin E2 release appears to come mainly from the subepithelial compartment and not from epithelial cells. Unlike its effect on anion secretion, phorbol 12,13-dibutyrate inhibition of sodium uptake by sodium-hydrogen exchange in isolated mature villus enterocytes is not inhibited by piroxicam. This study concludes that phorbol ester effects on anion secretion in chickens appear to be mediated by arachidonic acid metabolites most likely produced by cells of the lamina propria and submucosa, whereas phorbol ester effects on inhibiting upper villus cell sodium-hydrogen exchange may be a direct effect of phorbol esters.