Labeled and unlabeled prostacyclin and 6-keto-PGF(1α) were infused into healthy volunteers; urine was chromatographed on different systems including high pressure liquid chromatography. The peaks obtained by the latter method were derivatized to the methoxime methyl ester trimethyl silyl ether and analyzed by gas-liquid chromatography-mass spectrometry. After infusion of prostacyclin the following metabolites could be identified: dinor-4-keto-7,9,13-trihydroxy-prosta-11,12-enoic acid (20.5%), dinor-4,13-diketo-7,9-dihydroxy-prostanoic acid (6.8%), dinor-4,13-diketo-7,9-dihydroxy-prostan-1,18-dioic acid (19.7%), and 6-keto-PGF(1α) (14.2%), the in vitro hydrolysis product of prostacyclin. 6-Keto-PGF(1α) infusion resulted in the same metabolites with the relative amounts of 22.4, 5.4, 7.0, and 6.8%, respectively. Additionally, 6,15-diketo,13,14-dihydro-PGF(1α) (5.7%) could be identified. These data show that the metabolic pathway of prostacyclin involves hydrolysis to 6-keto-prostaglandin F(1α), subsequent β-oxidation, dehydrogenation at C-15, reduction of the double bond between C-13 and C-14, and ω-oxidation to the dicarboxyl metabolite. We conclude that dinor-4-keto-7,9,13-trihydroxy-prosta-11,12-enoic acid and dinor-4,13-diketo-7,9-dihydroxy-prostan-1,18-dioic acid represent the major urinary metabolites of prostacyclin in man. 6-keto-PGF(1α) is a minor urinary excretory product following the administration of prostacyclin or 6-keto-PGF(1α).