A major androgen-dependent urinary protein of male rodents, alpha 2u-globulin, has been shown to influence adenohypophyseal hormone release. In an attempt to elucidate the mechanisms of its action, we have examined several parameters of hypothalamic and pituitary function in adult male rats treated for 2 weeks with two injections daily of 0.75 mg alpha 2u-globulin and sacrificed 16 h after the last injection. This treatment led to an increase in plasma luteinizing hormone levels, a decrease in plasma prolactin levels, an increase in testosterone concentrations in both plasma and testicular tissue, and increases in testicular weights. The norepinephrine turnover in median eminence and anterior hypothalamus was increased in alpha 2u-globulin-injected animals, while the norepinephrine turnover in the remaining medial basal hypothalamus was reduced. alpha 2u-Globulin-treated animals had a significantly decreased dopamine turnover in the anterior hypothalamus, while in the medial basal hypothalamus the dopamine metabolism was increased. These data suggest that alpha 2u-globulin-induced changes in gonadotropin and prolactin secretion are mediated by changes in catecholamine metabolism in several hypothalamic regions. Increased testosterone secretion appears to be due to increased secretion of gonadotropins from the pituitary.