Estramustine phosphate, a nitrogen mustard derivative of estradiol used for the treatment of advanced prostatic cancer, was administered orally to man, rat and dog and the plasma concentrations of its unconjugated metabolites were determined by high performance liquid chromatography. In all species the drug was rapidly and completely dephosphorylated prior to reaching the peripheral circulation. In man and the rat, the 17-keto analogue of estramustine (estromustine) was the mamor metabolite found in plasma with considerably lesser amounts of estramustine itself. In the dog, significant concentrations of both estramustine and estromustine were present. Highly elevated levels of both estrone and estradiol, as a result of cleavage of the nitrogen mustard from the steroid, were observed in all species. Chronic administration of therapeutic doses of estramustine phosphate to man did not result in any apparent accumulation of circulating metabolites. The study suggests that the metabolite, estromustine, in addition to estramustine, may play an important role in the therapeutic efficacy of estramustine phosphate.