Nitric oxide synthase is the biosynthetic enzyme for the free radical neurotransmitter nitric oxide. Using an affinity-purified antiserum, nitric oxide synthase was found to be localized to peripheral ocular nerve fibers, related cranial ganglia, and the retina of the rat. In the eye, nitric oxide synthase-like immunoreactive peripheral nerve fibers were visualized mainly in the choroid and about limbal blood vessels. The anterior uvea was quite sparsely innervated, and the cornea was negative. Many principal neurons in the pterygopalatine ganglion were immunoreactive for nitric oxide synthase while very few cells stained in the superior cervical and trigeminal ganglia. Virtually all nitric oxide synthase-like immunoreactive pterygopalatine cells were also immunostained for vasoactive intestinal polypeptide; nitric oxide synthase also partially co-localized with neuropeptide Y in some of the neurons of this ganglion. Pterygopalatine ganglionectomy significantly reduced the number of peripheral nitric oxide synthase-like immunoreactive nerve fibers in the eye. A variety of immunoreactive retinal cells were seen. Most cells in the inner nuclear layer or ganglion cell layer corresponded morphologically to amacrine cells and displaced amacrine cells. Interplexiform cells and occasional faintly stained cells in the outer portion of the inner nuclear layer also were visualized. Nicotinamide adenine dinucleotide phosphate diaphorase histochemistry generally stained cells of similar distribution but did reveal somewhat more extensive localizations in peripheral ocular tissues, the ciliary ganglion, and the retina, compared with nitric oxide synthase immunohistochemistry. Nitric oxide synthase thus localizes to peripheral ocular nerve fibers, chiefly parasympathetic in nature and derived from the pterygopalatine ganglion, and to several cell types in the retina. Nitric oxide probably acts as a choroidal vasodilator of parasympathetic origin in the eye; the neuropeptide co-localizations in the pterygopalatine ganglion suggest complex neuromodulatory interactions. The retinal localizations imply potential neurotransmitter functions for nitric oxide in this tissue.