Photostimulation of retinal photoreceptors appears to inhibit reproductive activity in birds. In the present study, the involvement of serotonin and vasoactive intestinal peptide was investigated in relation to reproductive failure associated with retinal photostimulation. Hens at 23 wk of age were divided into six rooms equipped with individual cages. At 24 wk of age, three rooms were photostimulated (14L:10D) with white light (control). Three rooms had two parallel lighting systems, red (660 nm) and green (560 nm), which were both on during 6 h of the 14-h light period. Upon photostimulation, the red light was turned off after 6 h, and the green light was left on for a total of 14 h (Green). Five hens from each room served as controls, five hens were immunized against vasoactive intestinal peptide, and five hens received parachlorophenylalanine, an inhibitor of serotonin biosynthesis. Parachlorophenylalanine treatment increased reproductive performance and mRNA expression of GnRH-I, LH-beta and FSH-beta (P < 0.05) in the Green group to levels which did not differ from those of the White (control) group. Immunization against vasoactive intestinal peptide reduced plasma concentration and pituitary mRNA expression of prolactin but did not affect expression of gonadal axis genes. Collectively, the results suggest that retinal photostimulation inhibits the reproductive axis through serotonin and not through vasoactive intestinal peptide.