Labeled and unlabeled prostacyclin and 6-keto-PGF1 alpha 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-PGF1 alpha (14.2%), the in vitro hydrolysis product of prostacylin. 6-Keto-PGF1 alpha 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-PGF1 alpha (5.7%) could be identified. These data show that the metabolic pathway of prostacyclin involves hydrolysis to 6-keto-prostaglandin F1 alpha, subsequent beta-oxidation, dehydrogenation at C-15, reduction of the double bond between C-13 and C14, and omega-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-PGF1 alpha is a minor urinary excretory product following the administration of prostacyclin or 6-keto-PGF1 alpha.