This report describes a radioimmunoassay method for the measurement of unconjusted 16a-hydroxydehydroepiandrosterone (16a-DHEA) in plasma. The assay uses a specific anti16a-DHEA antiserum which cross reacted completely with dehydroepiandrosterone (DHEA) and to a lesser extent with androstenetriol and androstenediol. A chromatographic system, using celite microcolumns, separated 16a-DHEA from the cross reacting steroids prior to its measurement. Using this simple technique, we have measured 16a-DHEA accurately in small aliquots of plasma from newborns and adults of both sexes. The highest levels of 16a-DHEA were found in newborn infants with a mean value of 16.1 ng/ml. Pregnant women at term had a mean level of 3.2 ng/ml. The levels of 16a-DHEA were low in adults of both sexes with mean values of 0.66 and 0.87 ng/ml for male and female subjects respectively