The elimination of [H]prostaglandin E, in anaesthetized rats was studied by continuous intravenous or intraarterial infusions, producing steady-state concentrations at the level of endogenous prostaglandin E in mixed venous blood. Blood samples (0.5 ml) were collected from the carotid artery or the right atrium, respectively. The levels of [H]prostaglandin E were measured at different infusion time intervals and the H-labeled hydrophobic metabolites characterized. Cardiac output was estimated by a modification of the dye injection method, using I-labelled albumin as the marker. From the cardiac output and the rate of infusion, the fractional clearance of the lung and the systemic beds in the steady-state situation were estimated to 88.3 ± 3.2% and 54.1 ± 15.2%(mean ± S.D.), respectively. The hydrophobic metabolites were characterized chromatographically on Sephadex LH-20 columns, using synthetically prepared [C]prostaglandin metabolites as internal standards and markers. The identities of some metabolites were further established by derivative formation to a constant [H]/[C] ratio. The major metabolite was 15-keto-13,14-dihydro-[H]prostaglandin E, while 15-keto-[H]prostaglandin E and 13,14-dihydro-[H]prostaglandin E could not be demonstrated.