Immunocytochemical localization of Na+,K+-adenosine triphosphatase (ATPase) in fixed sections of rat retina was carried out using polyclonal antibodies to the highly purified enzyme and peroxidase-antiperoxidase and immunofluorescence techniques. In the photoreceptor layer of the rat retina, Na+,K+-ATPase immunoreactive sites were associated primarily with inner segments and only background activity was associated with the outer segments. This localization supports the proposed distribution of Na+,K+-ATPase in photoreceptor cells suggested by physiological studies. The Na+,K+-ATPase immunoreactive sites were also localized in the inner and outer plexiform layers, which are rich in synaptic contacts. The reticular pattern of immunostaining in those regions suggests that Na+,K+-ATPase immunoreactivity is associated with neuronal processes in those layers.