Normal colonocytes in culture produce prostaglandins both constitutively and in response to inflammatory stimuli. These highly purified proliferative cell populations were isolated from normal adult rabbit proximal and distal colon. Basal prostaglandin production ranged from 3.4 to 11.7 ng.15 min-1 x 10(6) cells-1. Cultures were incubated at 37 degrees C in the presence or absence of bradykinin or N-formyl-methionine-leucine-phenylalanine (FMLP) over concentrations from 10(-9) to 10(-5) M. In both distal and proximal colonocytes bradykinin stimulated a dose-dependent increase in prostaglandin E2 (PGE2) and 6-keto-PGF1 alpha, the stable metabolite of prostacyclin; production peaked at 10(-6) M. Proximal colonocytes responded to FMLP with a bell-shaped curve, with maximal stimulation of both PGE2 and 6-keto-PGF1 alpha occurring at 10(-8) M. Distal colonocytes responded variably to FMLP. Arachidonic acid also stimulated prostanoid production in a concentration-dependent manner, with maximal stimulation occurring at 100 microM. The full synthetic profile of prostanoid production was determined by labeling with [14C]arachidonic acid and by analyzing metabolites using radiochromatography on reverse-phase high-pressure liquid chromatography. Only PGE2 and 6-keto-PGF1 alpha were detected. A similar profile of labeled metabolites occurred when colonocytes were prelabeled with [14C]arachidonic acid and stimulated with bradykinin or FMLP. The degradative capacity of the colonocytes appeared very low. Colonocyte production of protective prostaglandins in response to luminal or other inflammatory stimuli may serve as a mucosal defense mechanism. Prostanoids so produced may also modulate the functions of colonocytes, surrounding cells, or both.