A radioimmunoassay using an antibody against llα,20α-dihydroxypregn-4-en-3-one-11-succinyl-BSA is described for the measurement of 20α-hydroxypregn-4-en-3-one in human plasma. Sephadex LH-20 column chromatography is used to separate progesterone from 20α-OHP. The sensitivity of the assay is 50 pg. The interassay and intra-assay coefficients of variation were 15% and 10%, respectively. Daily measurement of 20α-OHP throughout the normal menstrual cycle revealed levels of 0.41 ± 0.05 ng/ml throughout the follicular phase, followed by a slow rise after ovulation reaching peak levels of 3.4 ± 0.5 ng/ml in the midluteal phase. A constant ratio of 20α-OHP/P of 0.15 to 0.22 was found throughout the luteal phase. Levels in men were not significantly different from those in the early follicular phase of the normal menstrual cycle.