A mixture of 14C–squalene and 3Hdehydroisoandrosterone was injected into the umbilical circulation of a pregnant mare on the 250th day of gestation. The abdomen was closed and maternal urine was collected for 4 days. Steroid conjugates present in the urine were hydrolyzed and separated into neutral and phenolic fractions. From the phenolic fraction estrone, 17a–estradiol, equilin, equilenin, 17a–dihydroequilin and 17adihydroequilenin were isolated. Only the classical estrogens, estrone and 17a–estradiol were radioactive and contained both 3H and 14C, while the ring B unsaturated estrogens were devoid of any radioactivity. From the neutral fraction r e – labeled 3P–hydroxy–5a–pregnan–20–one, 5a–pregnane– 3P,20P–diol and 5a–pregnane–3P,20a–diol were isolated. These results, together with our previous findings, demonstrate that ring B unsaturated estrogens are formed independently of the classical pathway of biosynthesis and that a bifurcation occurs at a point prior to the formation of squalene. This is the first example of steroid biosynthesis through a squalene and cholesterol independent route. (Endocrinology92: 657, 1973)