Experiments were conducted to analyze the effects of growth hormone and testosterone in conjunction with the effects of neonatal sexual differentiation (via castration of males at days 2 or 11 of age and androgenization of females at day 3 of age) on thymic weight and thymosin-beta 4 concentrations in hypophysectomized rats (day 30 of age). Ten days post-hypophysectomy, hormonal treatments were initiated on males, male castrates, females, and androgenized females. Growth hormone (25 micrograms daily), testosterone propionate (100 micrograms/day), and the combination of the two hormonal treatments were administered for seven days, then thymic weights and blood samples were collected. Administration of growth hormone to hypophysectomized rats increased thymosin-beta 4 concentration in a dose-dependent manner, but injection of testosterone had no effect on thymosin-beta 4 concentrations. Testosterone treatment decreased thymic weights whereas growth hormone increased thymic weights. Hypophysectomized males had increased thymosin-beta 4 concentrations compared with female and neonatally-androgenized female rats. However, hypophysectomy eliminated any thymic weight differences between males and females. The data support a possible endocrine role for the thymus gland and thymic peptides in that they are integrated into the control and support of other endocrine systems. Although differences in thymosin-beta 4 concentrations were noted between males and females, sexual differentiation of the immune system was unaltered by neonatal castration of males or testosterone treatment of females and may indicate sexual differences in immune function are established in utero.