The major metabolites of prostacyclin (PG12) in human plasma have been determined after intravenous infusion of tritium‐labeled and unlabeled prostacyclin. Plasma was extracted and chromatographed. On high‐pressure liquid chromatography (HPLC), several radioactive peaks could be resolved. The major peak containing 41.6% of the radioactivity had the retention volume of authentic 6‐keto‐prostaglandin F1α(6‐keto‐PGF1α), the stable in vitro hydrolysis product of prostacyclin. When the material of this peak was derivatized to the methoxime methyl ester trimethylsilyl ether and analyzed by gas chromatography–mass spectrometry, the fragments m/z 508 and 598, which are characteristic of this derivative of 6‐keto‐PGF1αwere detected. A much smaller peak representing 6.6% of the radioactivity eluted from HPLC with the same retention volume as dinor‐6,15‐diketo‐13,14‐dihydro‐PGF1α. On gas chromatography–mass spectrometry this material resulted in the fragments m/z 527, 468, 437, and 347, which are characteristic for this prostanoid. Finally, 10.1% of the radioactivity with ions m/z 571, 481, 391, and 354 on mass spectrometric analysis could be identified as dinor‐6,15‐diketo‐13,14‐dihydro‐20‐carboxyl‐PGF1α. it is concluded that 6‐keto‐PGF1αrepresents the major breakdown product of prostacyclin in human plasma, in addition, dinor‐6,15‐diketo‐13,14‐dihydro‐PGF1αand its ω‐oxidized analog could be identified circulating metabolites.Clinical Pha