The tuberoinfundibular dopaminergic (TIDA) neurons projecting to the external zone of the median eminence arise in the dorsomedial and ventrolateral subdivisions of the arcuate nucleus. In cycling female rats these regoins contain only scattered enkephalin-immunoreactive (ENK-i) neurons some of which coexpress dopamine, detected by immunostaining for tyrosine hydroxylase (TH). The present immunocytochemical, in situ hybridization and retrograde-labeling studies show that each TIDA neuron of pregnant, pseudopregnant, lactating, and aged female rats contains ENK-like immunoreactivity and pro-ENK mRNA and projects to the hypophysial portal circulation. Ovariectomy of lactating and aged rats did not change ENK staining within TIDA neurons, suggesting that ovarian steroids do not play a critical role in the colocalization of ENK and dopamine. Since prolactin levels are elevated in each of these experimental animals, a possible role for prolactin in the induction of the ENK gene in TIDA neurons is suggested. Prolactin stimulates dopamine and its own secretion via a short-loop feedback mechanism. The sensitivity of this regulatory mechanism is altered in these experimental animals, resulting in elevated prolactin secretion. ENK, which has prolactin-releasing activity and is colocalized with dopamine, could mediate the positive short-loop feedback regulation and sustain elevated levels of prolactin in pregnant, pseudopregnant, lactating, and aged animals.