This study demonstrates that in addition to using the maturational steroid hormone 17,20β-dihydroxy-4-pregnen-3-one (17,20β-P) as a potent sex pheromone, the goldfish uses its sulfated metabolite 17,20β-dihydroxy-4-pregnen-3-one 20-sulfate (17,20β-P-20S). As measured by electro-olfactogram recording (EOG), the goldfish olfactory epithelium is extremely sensitive to both free and sulfated 17,20β-P (detection thresholds of approximately 10-12 and 10-11 M) but not to glucuronidated or phosphated 17,20β-P. Furthermore, both structure-activity and cross-adaptation studies using EOG indicated that 17,20β-P-20S and 17,20β-P are detected by different olfactory receptor sites, suggesting that these cues function as a distinguishable mixture. Finally, although the pheromonal activity of 17,20β-P-20S appears to be slightly less than that of 17,20β-P, it too stimulates gonadotropin release and sperm production in male goldfish. The precise function of 17,20β-P-20S remains somewhat enigmatic, however, because it does not appeal to enhance the actions of 17,20β-P and is released by a different mechanism. In any case, our findings clearly establish that a fish olfactory system can detect particular conjugated steroidal compounds in a highly specific manner and that naturally released conjugated steroids call function as components of a pheromonal mixture.