Pheromone-containing and pheromone-devoid samples of male sea lamprey (Petromyzon marinus) urine were analyzed for the concentrations of nine steroids [dehydroepiandrosterone (DHEA), testosterone (T), dihydrotestosterone (DHT), progesterone (P), androstenedione (A), estrone (E1), estradiol (E2), corticosterone (B), and cortisol (F)] by radioimmunoassay (RIA). Samples analyzed included native urine that had been enzymatically hydrolyzed with mixed β-glucuronidase/sulfatase. Values of the analyses were used to prepare solutions of the individual steroids for bioassay at concentrations which bracketed the urinary concentrations. Results show that only testosterone elicited a preference response in spawning-run female sea lampreys, and in concentrations three to four orders of magnitude greater than those found in active, unhydrolyzed male urine. The possibility that testosterone acts as a pheromone in this species is discussed.