Estradiol (E2) produces many-fold increases in the dopamine (DA) content of the anterior pituitary and also plays a role in the age-related increase in pituitary DA in female C57BL/6J mice. These studies address the following questions: (1) What are the time and dose characteristics of the E2-induced increase in pituitary DA and can other gonadal steroids – such as progesterone (P) and 5α-dihydrotestosterone – also influence pituitary DA? (2) Is the age-related increase in pituitary DA due entirely to an increase in the E2:P ratio seen in aging female mice, or can extra-ovarian factors also play a role? (3) Is the E2-induced (and therefore possibly the age-related) increase in pituitary DA secondary to an E2-induced increase in serum prolactin? In ovariectomized (OVX) mice, E2 implants increased pituitary DA in a time- and dose-dependent fashion. P implants administered to OVX mice simultaneously with E2 antagonized the E2-induced increase in pituitary DA. Daily injections of 5α-dihydrotestosterone given in conjunction with E2 implants had no effect on basal or E2-increased pituitary DA in OVX mice. Thus, E2 is the only gonadal steroid examined which increases anterior pituitary DA. In intact aging mice, P attenuates the age-related increase in pituitary DA, supporting the hypothesis that the increased ratio of E2:P secreted by the ovaries of aging female mice is responsible for the age-related increase in pituitary DA. However, at advanced ages, intact male mice also showed modest increases in anterior pituitary DA. Therefore, extra-ovarian age changes, perhaps age changes intrinsic to the pituitary, also play a minor role in the age-related increase in pituitary DA. E2 increases the secretion of prolactin, which can then feed back to increase secretion of DA from the tuberoinfundibular dopaminergic neurons, a source of pituitary DA. To determine if the E2-induced increase in pituitary DA was secondary to an E2-induced increase in serum prolactin, OVX mice were given pituitary grafts to increase serum prolactin independently of E2 treatment. The E2-induced increase of pituitary DA is not mediated, but can be enhanced by prolacti