Antibodies against the main urinary metabolite of PGF2alpha in the human, 5alpha, 7alpha-dihydroxy-11-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the omega position to bovine serum albumin prior to injection. The resuling antibodies did not distinguish between tetranor compounds varying only in structure at the omega carbon, and thus the assay could be used also for other metabolites of PGF2alpha, e.g. the main urinary metabolite in the guinea pig, 5alpha,7alpha-dihydroxy-11-ketotretranorporstanoic acid. Labeled ligands for the assays were prepared either in vivo by injection of [17, 18-3H]-PGF2alpha into humans after several days treatment with indomethacin, or in vitro by incubation of [17, 18-3H]-15-keto-13, 14-dihydro-PGF2alpha with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively. The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF2alpha; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment. The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.