Alpha 2 adrenergic agonists are used for controlling intraocular pressure (IOP) in the treatment of glaucoma. They have also been shown to be neuroprotective to retinal cells in a variety of injury models. Despite this significance, the localization of the three known alpha 2 adrenergic receptors has not been unequivocally established. The aim of this study was to determine the location of the three alpha 2 adrenergic receptors in ocular tissues using immunohistochemical techniques. New antibodies were generated and their specificity was determined using Western blotting and preadsorption. In the anterior segment of the eye alpha 2A immunoreactivity was located in the nonpigmented ciliary epithelium, corneal, and conjunctival epithelia. Alpha 2B staining was not apparent in these tissues. Alpha 2C immunostaining was present in the membrane of pigmented ciliary epithelium and corneal and conjunctival epithelial cells. In the rat retina, all three receptor subtypes were present but were differentially localized. Alpha 2A was present in the somata of ganglion cell layer and inner nuclear layer somas, alpha 2B was located in the dendrites and axons of most of the neurons as well as glia, while alpha 2C was present in the somata and inner segment of the photoreceptors. In human and monkey retinas, similar pattern of labeling for alpha 2A and 2B receptors were observed, while alpha 2B was additionally present in the membranes of many cell somata in addition to dendrites and axons. Alpha 2C labeling was much weaker but exhibited similar pattern to that observed in the rat. These data provide additional information on the location of the alpha 2 receptors in the anterior portion of the eye and present new information on their specific location in the retina. This offers insights into possible targets for adrenergic agonists in a therapeutic context.