Immunohistochemistry and in vitro autoradiographic techniques were used to investigate the localization of endothelin (ET)-like immunoreactivity and [125I]ET-1 binding sites in sections of ocular tissues of normal rabbits. ET-like immunoreactivity was localized to the corneal endothelium and the pre-epithelial layer of the cornea, which was specifically inhibited by addition of exogenous ET-1. Immunoreactivity for ET was also observed in the retinal ganglion layer and periocular fat tissues. These, however, were not inhibited in the presence of ET-1. Autoradiography showed binding sites for ET-1 in the iris and ciliary body. Emulsion-dipped slides of sections of rabbit eyes incubated with [125I]ET-1 showed specific labeling for binding sites over structures that were identified from serial sections stained with hematoxylineosin. These were the anterior stroma of the iris root, the walls of capillaries, the circular arteries, and the veins of the ciliary body and extraocular muscles. No binding sites were found in the cornea or conjunctiva. The difference in regional distributions of ET storage and binding sites in rabbit ocular tissue suggests that the peptide may have specific physiologic functions.