Plasma levels of 17,20β-dihydroxy-4-pregnen-3-one (17,20βOHP), which is involved in the regulation of spermiation in male salmonid fish, increase dramatically at the time of spermiation. To advance the understanding of the regulation of 17,20βOHP production during the spermatogenetic cycle in trout, we have studied the in vitro effect of gonadotropin type .II (GtH .II) and the precursor 17-hydroxy-4-pregnene-3,20-dione (17OHP) on the production of 17,20βOHP. The sensitivity with which testes secreted 17,20βOHP following stimulation with GtH .II was maximum during spermatogenesis. The addition of 17OHP (10 to 1600 ng ml-1) to the culture medium of testes fragments induced a significant and dose-related increase in 17,20βOHP secretion. Although the capacity to produce 17,20βOHP was not saturated by the 17OHP concentrations used, the conversion rate was highest for tested at an immature stage. As to the regulation of 17OHP, in vivo, a single injection of partially purified salmon gonadotropin (50 ng g-1 body weight) induced a significant increase in the circulating levels of 17OHP of immature males. In conclusion, the maximum sensitivity to GtH .II stimulation and the highest conversion rate of 17OHP to 17,20βOHP in vitro, occurred before the dramatic increase in the 17,20βOHP secretion observed in rainbow trout at the time of spermiation.