Quantitative analysis of metabolite levels is a useful approach to investigation of the in vivo synthesis of short-lived mediators such as prostacyclin (PGI2). In order to establish the basis for metabolite assays of PGI2, we have studied the fate of radiolabeled PGI2 administered to man. Three healthy male volunteers each received i.v. 11 beta-[3H]PGI2 at 4 ng/kg/min for 24 hr. A gradual increase in plasma radioactivity was observed throughout the infusion period, followed by a biphasic decline postinfusion (T 1/2 alpha, 53 min; T 1/2 beta, 246 min) suggestive of the presence of long-lived metabolites of PGI2 in the circulation. The recovery of radioactivity averaged 82% in urine and, in contrast to other species, only 4% in feces. Direct analysis of urine by high-pressure liquid chromatography revealed the presence of at least 16 compounds and documented their relative abundance. Ten compounds were subsequently identified by gas chromatography-mass spectrometry. All identified metabolites retained the 6-keto-prostaglandin F structure characteristic of PGI2 hydrolysis and were each formed in less than 10% yield from administered PGI2. Of interest was the finding that 6-keto-prostaglandin F1 alpha accounted for 5.9% of systemically administered 11 beta-[3H]PGI2. These results identify urine as the major route of human PGI2 metabolite excretion and also illustrate the utility of direct chromotographic analysis of urine in the elucidation of prostaglandin disposition in humans.