Lactation is a state of hyperprolactinemia resulting in part from suppressed tuberoinfundibular dopaminergic (TIDA) neuronal activity. The suckling stimulus contributes to this suppression despite the fact that the TIDA neurons are a potential site for PRL feedback to increase neuronal activity. This study examined the influence of PRL feedback and the suckling stimulus on tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine biosynthesis, during early and midlactation. On day 3 or 10 of lactation, rats were injected intracerebroventricularly with medium (control) or MMQ cells (200,000 cells), a PRL-secreting cell line. On day 6 of lactation, TH activity in the stalk-median eminence was increased 2- or 1.4-fold by MMQ cells or prior treatment with ovine PRL (oPRL; 4 mg/kg, sc), respectively. Removal of pups for 24 h increased TH activity 70% above levels in pup-exposed rats, and MMQ cells or oPRL caused an additional 60% increase. TH messenger RNA (mRNA) levels in the arcuate nucleus were increased 3-fold after removing the pups, but MMQ cells did not alter mRNA levels in either pup-exposed or pup-deprived dams. In contrast to early lactation, MMQ cells did not alter TH activity or mRNA levels in the pup-exposed dams on day 13 and only marginally increased enzyme activity in pup-derived dams. Circulating PRL levels were markedly reduced after removing pups. MMQ cells suppressed circulating PRL levels in both groups of dams on day 6 and in pup-deprived dams on day 13, but had no effect in pup-exposed dams at this time. In a second experiment, pup-exposed dams were injected with bromocriptine, a dopamine agonist, and killed after 4 or 12 h on day 5 or after 12 h on day 12. In some rats, PRL was replaced by injecting oPRL simultaneously with and 8 h after bromocriptine treatment. On day 5 of lactation, bromocriptine reduced and oPRL restored TH activity, whereas on day 12, oPRL was not able to reverse the effect of bromocriptine. These data indicate that the suckling stimulus suppresses TH activity and gene expression in the TIDA neurons in pup-exposed dams. The high endogenous PRL levels associated with the suckling stimulus may activate TH activity in TIDA neurons during early lactation. However, the responsiveness of the TIDA neurons to PRL feedback is attenuated by day 13 of lactation.