The effect of 13-hydroperoxyoctadecadienoic acid (13-HPODE) on the synthesis of prostaglandins (PGs) was examined in rabbit kidney medulla microsomes. Medulla microsomes were incubated with arachidonic acid in 0.1 M-Tris/HCl buffer (pH 8.0) containing reduced glutathione and hydroquinone and the PGE2, PGF2a and PGD2 formed were measured by high-pressure liquid chromatography using 9-anthryldiazomethane for derivatization. Under our incubation conditions rabbit kidney medulla was found to produce mainly PGE2. The addition of 13-HPODE inhibited the production of all three PGs to a similar extent. 13-Hydroxyoctadecadienoic acid did not suppress the formation of PGs, indicating the requirement of the hydroperoxy moiety for the inhibitory effect of 13-HPODE on PG formation. Experiments utilizing the native fatty acid linoleic acid and tert-butyl hydroperoxide suggested the importance of the fatty acid-derived hydroperoxide in PG synthesis by kidney medulla. We conclude that 13-HPODE is an inhibitor of renomedullary cyclooxygenase and may have functional effects within the kidney.