The peripheral plasma content of the pulmonary metabolite, 13,14-dihydro-15-keto-prostaglandin E2, reflects prostaglandin E2 (PGE2) biosynthesis and disposition in vivo more reliably than the peripheral plasma content of PGE2 itself. However, the chemical instability of 13,14-dihydro-15-keto-PGE2 and the multiple fates of its degradation products hinder the development of quantitative assays for the metabolite. We eliminated these problems with an indirect approach. At pH 10.5, intact 13,14-dihydro-15-keto-PGE2 and its degradation products convert uniformly into 11-deoxy-13,14-dihydro-15-keto-11 beta, 16 xi-cyclo-PGE2. We developed a radioimmunoassay with a sensitivity of 12-pg for this analytically suitable bicyclic derivative. The accuracy, precision and sensitivity of the method permitted its application to certain intractable problems. For example, plasma levels of the pulmonary metabolite can be quantitated and used as an index of the pharmacokinetic disposition of PGE2 because its metabolite forms almost instantaneously and completely, in vivo. In dogs, there was a dose-dependent increase in plasma 13,14-dihydro-15-keto-PGE2 concentrations after bolus i.v. injections of PGE2. Ten minutes after administration of 30, 10 or 3 micrograms of PGE2 per kg, its plasma metabolite concentrations were 34.1 +/- 8.4, 6.8 +/- 0.6 and 2.0 +/- 0.2 ng/ml (mean +/- S.E.M., n = 4). the metabolite disappeared from the circulation rapidly with half-lives of 9.9 +/- 0.2, 9.2 +/- 0.3 and 7.8 +/- 0.8 min (mean +/- S.E.M., n = 4) for the respective doses noted above. Similar studies with PGE1 were possible because of the predictable cross-reaction between the bicyclic derivatives of 13, 14-dihydro-15-keto-PGE2 and 13,14-dihydro-15-keto-PGE1. Because the latter compound is not normally present in mammals, measurements of endogenous 13,14-dihydro-15-keto-PGE2 are still accurate. Basal concentrations of 48 +/- 31 pg/ml (mean +/- S.D., n = 15) in human plasma concur with concentrations measured by gas chromatography/mass spectrometry.