The role of the serotoninergic system in the control of prolactin (PRL) secretion has been studied in prepubertal male rats. Serum PRL concentration was measured in 16-day-old male rats at different times after the administration of 5-hydroxytryptophan (5-HTP), a precursor of serotonin (5-HT) synthesis, alone or in combination with fluoxetine, a specific inhibitor of 5-HT uptake; DL-p-parachlorophenylalanine (PCPA), an inhibitor of 5-HT synthesis; and 8-hydroxy-2-(di-n-propylamino)tetralin (8-OH-DPAT), a selective agonist of 5-HT1A receptors. Also, serum PRL concentration and pituitary content were measured after 5-HTP administration in castrated males implanted with silastic capsules containing testosterone or 5 alpha-androstane-3 alpha,17 beta-diol (alpha-diol). We found that: the reduction in serotoninergic activity after PCPA administration did not modify serum PRL concentrations; the stimulatory effect of 5-HTP on PRL secretion was not observed before day 16; the effects of 5-HTP or 5-HTP and fluoxetine were similar in intact and orchidectomized males; a significant increase in PRL secretion took place after 8-OH-DPAT administration; the duration of the stimulatory effect of 5-HTP increased after alpha-diol treatment; and pituitary PRL content increased after 5-HTP injection in intact males and decreased in castrated males treated with testosterone or alpha-diol. Therefore, we conclude that: the PRL-releasing effect of 5-HTP remains after orchidectomy; activation of 5-HT1A receptors may mediate, at least partially, the effect of 5-HTP; testosterone and alpha-diol affect the duration of PRL release after 5-HTP administration differently; and testicular factors other than androgens might be involved in the effects of 5-HTP on PRL pituitary accumulation.