Steroid sulfotransferase activity has been assayed in cytosol extracts obtained from the male hamster reproductive tract. Dehydroisoandrosterone and desmosterol were used as substrates in the presence of phosphoadenosine phosphosulfate-35S as sulfate donor. No significant sulfotransferase activity was found in the testis. In the epididymis, a severalfold increase in activity was found in the tissue from the caput to the caudal regions. A lower but significant activity was detected in the vas deferens. The enzyme appears to be secreted into the luminal fluid while little activity is associated with the spermatozoa. This increase in activity along the epididymis is undoubtedly responsible for the accumulation of sterol sulfates reported previously. In view of the fact that sterol sulfates are potent and specific inhibitors of acrosin, as reported for the porcine and confirmed herein for hamster acrosin, the epididymal production of steroid and sterol sulfates may represent a protective mechanism against the premature release of proteolytic activity within the male reproductive tract.