Hypothalamic neuropeptide Y (NPY) and proopiomelanocortin (POMC)-derived peptides located in the arcuate nucleus (ARC) have been postulated to be good candidates to play a modulatory role during lactation. In the present study, we first quantified, by in situ hybridization, lactation-induced changes in NPY and POMC gene expression throughout the ARC. In a second phase of the study, we attempted to determine whether any relationship exists between neuropeptide gene expression and the suckling stimulus itself. For this, we used experimental groups of animals submitted to suppression of the suckling stimulus by removal of pups and the subsequent restoration of the suckling stimulus by the return of the litter. Since lactation is characterized by an estrogen-deficient status [15], we attempted using ovariectomized (2 or 21 days) diestrous females to describe the changes in NPY gene expression observed during lactation. Since the suckling stimulus induces a strong prolactin (PRL) release, we completed this study by using an intravenous injection of PRL antiserum in order to discriminate the effects of PRL per se on the observed suckling-induced changes in neuropeptide gene expression. Freely nursing lactating females exhibited a large increase in NPY mRNA expression as compared to diestrous females (10.10 ± 0.50 vs. 4.51 ± 0.35). After suppression of the suckling stimulus by removal of pups, this increase intensified during short-term suppression of 16 h (15.37 ± 0.67) and was reversed following long-term suppression of 36 h (12.35 ± 0.61). Ovariectomized diestrous animals showed significant changes in NPY mRNA expression as compared to lactating females (5.25 ± 0.42 vs. 10.10 ± 0.50). Lactating females submitted to PRL immunoneutralization by PRL antiserum showed a slight increase in NPY mRNA expression as compared to non-injected lactating females (13.75 ± 0.51 vs. 12.95 ± 0.59). Freely nursing lactating females showed a decrease in POMC mRNA expression (8.27 ± 0.33) whereas suppression of suckling by removal of the pups (9.52 ± 0.45) resulted in a return to diestrous POMC mRNA levels (10.77 ± 0.36). We showed that restoration of suckling by the return of the litter induced an increase in POMC gene expression (12.55 ± 0.66). By lowering circulating levels of PRL with PRL antiserum after restoration of suckling, we observed a decrease in POMC mRNA expression (9.81 ± 0.46). Results of this study showed that the increase in NPY mRNA in the medial ARC during lactation did not appear to be due either to gonadal steroid-deficient status or to the suckling-induced hyperprolactinemia. If freely nursing lactating females showed a moderate decrease in POMC gene expression, restoration of the suckling stimulus by return of the pups provoked an increase in POMC gene expression which seemed to depend on high endogenous l