The roles of prolactin (PRL) and the ovarian hormones, estradiol and progesterone, in the control of tuberoinfundibular dopaminergic neurons of aged female rats were investigated. The in situ molar activity of tyrosine hydroxylase (TH) in neurites of these neurons was assayed by measuring the rate of accumulation of L-dihydroxyphenylalanine in the median eminence following the administration of a L-dihydroxiphenylalanine decarboxylase inhibitor. The TH mass was measured by an immunoblot assay using rat TH as the standard. Pituitary implants in aged ovariectomized animals resulted in a significant increase in the median eminence of both the mass and in situ molar activity of TH. When circulating PRL of aged rats was neutralized by administration of antiserum against rat PRL, the activity of TH was reduced significantly compared to that of animals treated with preimmune serum. In aged ovariectomized rats treated with both estradiol and progesterone, the in situ molar activity of TH increased significantly compared to animals treated only with the solvent vehicle, estradiol, or progesterone. The stimulatory effect of estradiol and progesterone appeared to be mediated through a mechanism that did not involve PRL, since neutralization of circulating PRL failed to prevent an increase in TH activity in estradiol-progesterone-treated animals. None of these treatments affected the in situ activity of TH in the superior cervical ganglion. We conclude that PRL as well as combined estradiol-progesterone treatment have important roles in the maintenance of TH activity in aged tuberoinfundibular dopaminergic neurons.