In prepubertal mice, subcutaneous thymulin injection before equine chorionic gonadotrophin (eCG) treatment simulates ovulation; seemingly, the thymulin could be acting at the hypothalamus-pituitary axis level. Objective: This study was designed to analyze the effects of injecting thymulin into the hypothalamus or pituitary on induced ovulation of prepubertal mice. Method: Female mice, 19 days old, were anesthetized with ether and injected with saline solution or thymulin into the anterior or medial hypothalamus or the pituitary and treated with eCG when 20 days old. The ova shed were counted and serum concentrations of 17β-estradiol were measured. In the ovaries, the morphometrical analysis was performed and the atresia evaluated. Results: Ether anesthesia treatment blocked eCG-induced ovulation in almost all animals. Mice anesthetized and treated with eCG and gonadotrophin-releasing hormone (GnRH) or human chorionic gonadotrophin (hCG) ovulated a full quota of ova. Injecting saline solution into the anterior or medial hypothalamus or the pituitary did not reduce the blocking effects of ether anesthesia on induced ovulation, but the incidence of atretic follicles was higher. Injecting thymulin directly into the anterior hypothalamus did not restore ovulation, nor diminish the number of atretic follicles. In contrast, injecting thymulin into the medial hypothalamus restored the ovulation ratio and decreased the percentage of atretic follicles. Similar results were obtained by injecting thymulin into the pituitary, though thymulin treatment in the pituitary resulted in a higher number of ova shed and lower follicular atresia. Conclusion: The present results suggest that thymulin acts at the medial hypothalamus level, facilitating the release of GnRH and at the pituitary level regulating gonadotrophin release.