A method is described for the differential extraction of unconjugated androgens (testosterone and dihydrotestosterone) and glucuronidated androgens (androstane-3α,17β-diol glucuronide and androsterone glucuronide) from human serum using solid-phase, gravity-flow extraction columns. In this method, 100-μL aliquots of serum are loaded onto the normal-phase columns, unconjugated androgens are eluted with ethyl ether, and glucuronides are eluted with ethyl ether containing 2% acetic acid. Glucuronide eluates are washed with 1% aqueous acetic acid to remove cross-reacting steroid sulfates. Assays of sera for the four steroids were performed using standard radioimmunoassay methodology, except for androsterone glucuronide. This steroid was assayed with a novel radioimmunoassay method that employs a tritiated, unconjugated androsterone tracer and an antidehydroepiandrosterone sulfate antiserum. The new method is well suited for the assay of conjugated and unconjugated steroids in large numbers of specimens, particularly where the sample volume is limited.