Several dopamine-related neurochemical and behavioral responses are influenced by the time of day. The light-dark shift is a major zeitgeber for various functionally important hypothalamic monoaminergic systems. However, these influences are modulated by reproductive state and by reproductive experience (RE) in females. Early pregnancy in rodents generates diurnal and nocturnal prolactin surges that are reduced in intensity in a second pregnancy. Dopamine (DA) is a major inhibitory factor of prolactin synthesis and secretion. Other neurotransmitters such as serotonin (5HT) and norepinephrine (NE) can modulate prolactin secretion as well. Previous works have demonstrated that RE induces changes in central concentrations of both dopamine and serotonin. In addition, RE modulates the responses of both dopaminergic and serotoninergic nerve terminals. The present investigation was designed to examine the possible effects of RE on hypothalamic concentrations of DA, NE, 5HT and their major metabolites homovanillic acid (HVA), 3-4-dihydroxyphenyl acetic acid (DOPAC), 3-methoxy mandelic acid (VMA) and 5-hydroxyindole 3-acetic acid (5HIAA), respectively. These parameters were measured in pregnant rats during the light-dark shift and the prolactin surges. Primi- and multigravid rats were sacrificed on the 7th-8th day of pregnancy between 1700 and 1900 h (light-dark shift and diurnal prolactin surge) or 0200 and 0400 h (nocturnal prolactin surge), and hypothalamic concentrations of DA, NE and 5HT and their metabolites were measured by high performance liquid chromatography coupled to an electrochemical detector (HPLC-ED). Trunk blood was collected and serum prolactin measured by radioimmunoassay. The prolactin surge was confirmed and multigravid rats showed significantly lower serum prolactin levels as compared to primigravid rats between 0200 and 0400 h. During the light-dark shift DA and NE concentrations increased while DOPAC/DA, HVA/DA and 5HIAA/5HT ratios decreased in multigravid rats compared to primigravid rats. Except for 5HIAA/5HT, these differences were not observed during the prolactin nocturnal surge. These results suggest that a previous reproductive experience induces central functional changes during pregnancy which are expressed differently according to the time of day.