The 5,7-dienes, 3β-hydroxy-5,7-pregnadien-20-one and 3β-hydroxy-5,7-androstadien-17-one were extracted from fetal horse gonads and purified by solvent partition, thin-layer chromatography and high performance liquid chromatography. The isolated steroids were identified by comparison with the synthetic steroids using ultraviolet and mass spectroscopy and by gas chromatography–mass spectroscopy. The identification of these compounds as endogenous steroids, together with the data on their biosynthesis reported previously, support the proposal that in the fetal horse gonad there is a 5,7-diene pathway biosynthesizing 3β-hydroxy-5,7-androstadien-17-one, which is the proposed precursor for equilin in the placenta.