5alpha-pregnane-3,20-dione was isolated from pooled pregnant mare serum using Sephadex LH-20 column chromatography and identified by the use of radioimmunoassay, gas-liquid chromatography and gas-liquid chromatography-mass spectrometry analyses. 5beta-pregnane-3,20-dione was not cross-reactive with the radioimmunoassay system and was not detected by gas-liquid chromatography. Peripheral blood levels of progesterone and 5alphs-pregnane-3,20-dione were determined by radioimmunoassay in four Quarter Horse mares for the first 150 days of gestation. Progesterone and 5alpha-pregnane-3,20-dione declined from a range of 6 to 15 ng/ml at day 10 to a range of 2 to 8 ng/ml at day 40. After day 40 there was an increase in progesterone and 5alpha-pregnane-3,20-dione concentration. Toward the end of 150 days of gestation progesterone tended to decline; whereas, 5alpha-pregnane-3,20-dione levels were maintained or increased to levels as high as 35 ng/ml. Neither source nor function of 5alpha-pregnane-3,20-dione is known.