This report describes a radioimmunoassay method for the measurement of unconjugated 17-hydroxypregnenolone (17-5δ-P) in plasma. Utilizing a highly specific 17-5δ-P 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 plasma levels of 17-5δ-P obtained by this method agreed well with levels observed in adult men and women, using two other techniques. Compared with the latters, however, the present method offered a tenfold greater sensitivity and a better precision.