Prostaglandin synthesis by bovine retinal pericytes was investigated using high pressure liquid chromatography to separate and identify 3H-labeled prostaglandins released from 3H-arachidonic acid labeled pericyte monolayers. A dominant peak activity corresponding to 6-keto-PGF1 alpha was observed. This peak was eliminated when monolayers were pretreated with cyclooxygenase inhibitors and was augmented when monolayers were stimulated by the calcium ionophore A23187. Suspensions of pericytes and the cell-free media of monolayers incubated with arachidonic acid inhibited adenosine diphosphate-, collagen-, and arachidonic acid-stimulated platelet aggregation in a bioassay for prostacyclin-like activity. This inhibitory activity was unstable at room temperature. Cultures of 7.5 to 10 x 10 (5) pericytes (7th passage near-confluence) released nanogram quantities of 6-keto-PGF1 alpha as measured by radioimmunoassay. These results are evidence that prostacyclin is the main prostaglandin synthesized by bovine retinal capillary pericytes in culture. Pericytes may influence the microcirculation via their production and release of this potent vasoactive arachidonic acid metabolite.