Serum concentrations of prolactin and biochemical estimates of impulse traffic in dopaminergic neurons (neuronal ‘activity’) were compared in rats on the second day of diestrus and in 12-day postpartum lactating rats that were either suckled or were nonsuckled (pup-deprived). Indices of the activities of tuberoinfundibular, tuberohypophyseal and nigrostriatal dopamine (DA) neurons were obtained by measuring the α-methyltyrosine-induced decline of DA concentrations, and the accumulation of dihydroxyphenylalanine (dopa) after the administration of an inhibitor of aromatic L-amino-acid decarboxylase in the median eminence, posterior pituitary and striatum, respectively. Biochemical indices of tuberoinfundibular DA neuronal activity and the content of DA in the anterior pituitary gland were lower in pup-deprived lactating rats than in diestrous controls, and were further reduced by suckling. These changes appeared to be specific for tuberoinfundibular DA neurons since biochemical estimates of tuberohypophyseal and nigrostriatal DA neuronal activity were the same in all three groups of animals. Following pup removal serum prolactin concentrations in the dam decreased to basal levels within 4 h and remained low for at least 72 h. The rate of dopa accumulation in the median eminence of lactating rats increased 4 h after pup removal, but remained significantly lower than diestrous values for 72 h. These results indicate that an acute suckling stimulus activates a neuronal circuit which causes a prompt decrease in tuberoinfundibular DA neuronal activity. This decrease may play a role in the suckling-induced increase in prolactin secretion. Independent of these acute changes, the ‘basal’ activity of tuberoinfundibular DA neurons is reduced in lactating rats. This decrease appears to result from the lack of responsiveness of these neurons to the stimulating actions of prolactin. This is supported by the fact that the rate of dopa accumulation in the median eminence was increased in diestrous animals 12 h after the intracerebroventricular injection of prolactin or 16 h after the administration of haloperidol (which increases serum prolactin concentrations) but these treatments were without effect in lactating animals. The decreased responsiveness of tuberoinfundibular DA neurons in lactating rats to prolactin may contribute to the ability of the anterior pituitary gland in these animals to maintain a high secretion rate of this hormone.