The onset of spawning behavior in male salmonids can be closely correlated to circulating levels of the progestin 17 alpha,20 beta-dihydroxy-4-pregnen-3-one (20-P). However, evidence that 20-P plays a causal role in the development and maintenance of spawning behavior in male salmonids is largely lacking. The purpose of this study was to investigate the effect of 20-P implants on the spawning behavior in castrated rainbow trout in the presence of a sexually active female. Mature rainbow trout were placed in five experimental groups: sham-operated and castrated implanted with either Silastic capsules containing 20-P, 11-ketoandrostenedione (11-KA), 11-KA + 20-P or empty capsules. Five to 6 weeks after implantation, all males were tested individually in a stream channel in the presence of a nesting ovulated female. Following behavioral observations, blood samples were taken from all tested males. Implants of 20-P and 11-KA resulted in increased plasma levels of 20-P and 11-ketotestosterone, respectively. Sham males exhibited the strongest behavioral response, both in the frequency of quiver bouts and attention toward the female. Castrated controls and 11-KA males showed no quiver response and reduced attention toward the female, while the behavioral responses shown by the 20-P and 20-P/11-KA males were intermediate. The results indicate that 20-P, but not 11-androgens, plays a significant role in the stimulation of spawning behavior in male rainbow trout.