A commercially available antidihydrotestosterone antiserum was used for the direct radioimmunoassay of androstenediol-3-sulfate (ADS) in human serum. Aliquots of 1 or 2 microliter male serum (mean age of 40 subjects, 38.2 +/- 5.0 years) were diluted and extracted with ethanol for assay. The tracer, [7-3H]ADS, was prepared by sodium borohydride reduction of [7-3H]dehydroepiandrosterone sulfate (DS). Significantly cross-reacting steroids were testosterone, DS, androsterone sulfate, and epiandrosterone sulfate, which combined to produce a mean overestimation of ADS of 4.3 micrograms/dl in male serum. Mean serum ADS was 23.6 +/- 10.0 micrograms/dl (SD) in 20 fresh-frozen sera versus 28.4 +/- 9.7 micrograms/dl (SD) in 20 long-term (24.4 +/- 1.2 years) frozen specimens, showing stability on long-term frozen storage. Androstenediol-3-sulfate also showed a strong correlation with serum DS (r = 0.75). The possible physiologic significance of ADS is discussed, particularly in terms of the known estrogenicity of unconjugated androstenediol.