This work was undertaken to study the effects of thymostimulin on the pituitary-ovarian axis of young female rats, and to elucidate possible prolactin-mediated effects. 30-day-old rats implanted with an ectopic pituitary from a littermate male donor under the kidney capsule or sham-operated animals were injected with thymostimulin or vehicle for 2 and 8 days. Chronic thymostimulin treatment to sham-operated rats increased plasma prolactin and estradiol levels, and decreased gonadotropin levels. Thymostimulin administration to pituitary-grafted rats did not further modify the increase in plasma prolactin and the decrease in plasma luteinizing hormone levels induced by the presence of an ectopic pituitary, suggesting a hypothalamic and/or hypophysial site of action of the thymic fraction. An increase in plasma estradiol and progesterone levels was observed with the in vivo thymostimulin treatment, although differential in vitro effects on estradiol responses to human chorionic gonadotropin in the various groups studied were observed. These effects seem to be mediated through the changes in plasma prolactin and gonadotropin concentrations observed. The above-mentioned changes induced by the thymic fraction on plasma estradiol and progesterone levels might counteract prolactin effects on the thymus. These data suggest that thymostimulin treatment of young female rats changes hypophysial and sex hormone secretion, as well as the response of the ovaries to human chorionic gonadotropin, probably modifying the normal process of sexual maturation.