This report describes a radioimmunoassoy method for measurement of unconjugated dehydroepiandrosterone (DHEA) in plasma. Utilizing a highly specific DHEA antiserum combined with a one step celite microcolumn chromatographic system, we have measured this steroid accurately in small aliquots of plasma from both male and female subjects. The mean value for both male and nonpregnant females was 2.7 ng/ml. The values for third trimester pregnant females ranged from 0.5 ng/ml to 12.5 ng/ml. When plasma DHEA was measured over a complete menstrual cycle, the levels were higher during the early and latter parts of that cycle than at midcycle. One male subiect, studied over a 24 hour period for diurnal variation, demonstrated highest DHEA levels during the early morning.