Photostimulation (PS) of turkeys increases the number of hypothalamic vasoactive intestinal peptide (VIP)-immunoreactive neurons, the number of anterior pituitary VIP binding sites, and prolactin (PRL) secretion. Serotonin (5-HT) was recently shown to stimulate PRL secretion through VIP. This study tested the hypothesis that 5-HT's ability to induce PRL secretion is mediated by reproductive status and/or photoperiod in normally cycling turkey hens. Initially, saline or 5-HT was infused into the third ventricle of nest-deprived, previously incubating (ND) hens for 60 min at rates of 0.1, 1.0, or 10 nmol/min. The results led to use of the 10 nmol/min infusion rate for the remaining 5-HT infusions. Next, 5-HT was infused into short-day (SD;6), laying (6), ND (5), and photorefractory (P/R;6) hens. Plasma PRL was elevated in all groups except for the SD hens (P < 0.05). In Experiment 3, VIP was infused into the median eminence of SD (6), laying (5), and P/R (5) hens, increasing circulating PRL levels in all three groups (P < 0.05). Finally, SD hens were photostimulated for 0, 3, or 10 days and then infused with 5-HT. Only the birds which were photostimulated for 10 days exhibited elevated plasma PRL (P < 0.05). In conclusion, PS regulates PRL secretion at the hypothalamic level and more than 3 days of PS are required for 5-HT- ergic stimulation of PRL secretion.