Despite its name, Vasoactive Intestinal Peptide (VIP), a 28-amino acid peptide, is widely distributed in the eye where it is thought to play a physiological role, particularly in aqueous humor dynamics or retinal neurotransmission. Localization and pharmacological properties of VIP binding sites were investigated in eyes from albino rabbit and rat using an in vitro autoradiographic method. 125I-VIP was used as ligand and unlabelled VIP was used to displace labelled VIP. Autoradiograms were generated by apposing the slides to 3H-Ultrofilm or autoradiographic emulsion and analysed using an image analysis system. Specific binding represented about 85% of total binding. Kinetic studies showed that equilibrium was reached after 140 min incubation at room temperature. Biochemical investigations demonstrated that 125I-VIP bound to a population of sites with high affinity (Kd = 2.95 +/- 0.5 nM). Inhibition of 125I-VIP binding with VIP and related peptide gave a rank order of potency: VIP greater than peptide histidine isoleucine greater than secretin greater than human growth hormone-releasing factor, glucagon, VIP1-14, VIP14-28. In both species, specific binding were found in conjunctiva, iris, ciliary processes, choroid and retina. Quantitative analysis of autoradiograms revealed that the highest densities of binding sites were localized in the ciliary epithelium in rabbits and in the inner retina in rats.