The localization of the messenger RNA (mRNA) encoding vasoactive intestinal peptide/peptide histidine isoleucine (VIP/PHI) in the rat eye was studied by in situ hybridization histochemistry using a synthetic 35S-labeled oligodeoxyribonucleotide. Among the layers of the retina, specific labeling was found in the soma of some cells in the innermost lamina of the inner nuclear layer. Occasionally, labeled cells were also present in the ganglion cell layer. No specific labeling was detected in any ocular structures other than the retina, including the cornea, iris, ciliary body, choroid, choriocapillaris, optic nerve and sclera. We also examined VIP/PHI mRNA expression in the suprachiasmatic nucleus (SCN) of the hypothalamus, which is the pacemaker of circadian rhythms and synchronizes them with the environmental light-dark cycle. High labeling was found in the ventrolateral part of the nucleus, which is the site of most projections from the retina. Monocular enucleation of rats at birth resulted in an increase of VIP/PHI mRNA expression in SCN ipsilateral to the removed eye after postnatal day 60. The discrete pattern of hybridization for VIP/PHI mRNA indicates that these two peptides are synthetized in rat retina and SCN and suggests that they may play a key role in the photic entrainment to the SCN regulating various circadian rhythms.