An initial experiment was conducted to analyze the effects of hypophysectomy (Days 5-30 post hypophysectomy) on thymic weight and thymosin-beta 4 concentrations. After hypophysectomy, thymic weight and thymosin-beta 4 concentrations decreased with time (5, 10, 15, and 30 days post hypophysectomy) and by 30 days post hypophysectomy were reduced by 50%. Exp. II investigated the effects of early sexual differentiation on thymic weight and thymosin-beta 4 concentrations in pituitary intact and hypophysectomized male, female, nonmasculinized rats (via castration of males at Day 2 or 11 of age and androgenized female (at Day 3 of age) rats. Rats were hypophysectomized at 30 days of age and, at 17 days post hypophysectomy, the thymus gland was dissected and weighed. Serum was collected for thymosin-beta 4 analysis. At 47 days of age, males have a greater thymic weight when compared to females (p less than 0.01) but hypophysectomy eliminated any sex differences and significantly reduced thymic weights (p less than 0.05). Hypophysectomy did produce increased thymosin-beta 4 concentrations across all treatments (p less than 0.01) and is considered to be a combination response of decreased gonadal function via lack of pituitary hormonal stimulation (luteinizing hormone and follicle stimulating hormone) and lack of gonadotrophic effects on the thymus gland and its secretory capacity. Hypophysectomized males and Day-2 castrate males had increased thymosin-beta 4 concentrations when compared to hypophysectomized females and neonatally androgenized females (p less than 0.05). Differences of thymic weights between male and female rats during prepubertal development are probably related more to presence or absence of gonads, gonadal steroids, and response of the thymus to pituitary secretagogues rather than to neonatal sexual differentiation of thymic function and secretion of thymosin-beta 4.