[3H]Equilin [3H-labeled 3-hydroxy-1,3,5(10), 7-estratetraen-17-one] was administered iv to a pregnant mare in the 10th month of gestation. Maternal urine was collected for 3 days, and blood samples were taken 35 min and 3, 6, 12, and 24 h after the injection. The half-life of the disappearance of radioactivity from the blood was approximately 2.5 h. Over 90% of the administered dose was excreted in the first 24 h. The urine was extracted, hydrolyzed, and fractionated. The bulk of the radioactive material (75%) was present in the phenolic sulfate fraction from which radiochemically pure equilin, equilenin [3-hydroxy-1,3,5(10),6,8-estrapentaen-17-one], 17 alpha-dihydroequilin [1,3,5(10), 7-estratetraen-3,17 alpha-diol], 17 beta-dihydroequilin [1,3,5-(10,7-estratetraen-3,17 beta-diol], 17 alpha-dihydroequilenin [1,3,5(10),6,8-estrapentaen-3,17 alpha-diol], and 17 beta-dihydroequilenin [1,3,5(10),6,8-estrapentaen-3,17 beta-diol] were isolated and identified. Except for equilenin, the above-named steroids were also isolated and identified from the glucuronide fraction. Along with these estrogens, the two classical estrogens, estrone and 17 alpha-estradiol, were also isolated, but both of these estrogens were devoid of any radioactivity. These results indicate that 1) the B ring unsaturated estrogens are not metabolized to the B ring saturated estrogens (classical estrogens), 2) all of the B ring unsaturated estrogens isolated and identified from the pregnant mare's urine are metabolites of equilin, 3) the major metabolite of equilin excreted in the urine was equilin sulfate, 4) from the specific activity of the isolated equilin sulfate and the amount of [3H]equilin injected, the secretion rate of equilin was calculated to be 96 mg/24 h, and 5) the major reduced metabolites of equilin are the biologically less active 17 alpha-reduced products.