We have used isolated embryonic photoreceptor cells as a model system with which to examine the mechanisms responsible for the development and maintenance of asymmetric Na+,K+-ATPase (ATPase) distribution. Photoreceptor precursors, which appear round and process free at culture onset, develop structural and molecular properties similar to those of photoreceptor cells in vivo. ATPase, recognized by an anti-ATPase antibody, is distributed over the entire surface of round photoreceptor precursors. As the cells develop, ATPase becomes progressively concentrated in the inner segment (where it is found in cells of the intact retina). This phenomenon occurs in cells developing in the absence of intercellular contacts. The development of ATPase polarity correlates with a decrease in the fraction of ATPase molecules that are mobile in the membrane (as determined by fluorescence photobleaching recovery), as well as with an increase in the fraction of ATPase that remains associated with the cells after detergent extraction. The magnitudes of the mobile ATPase fractions agree well with those of the detergent-extractable fractions in both the immature and developed photoreceptors. The distribution of alpha spectrin and ATPase-immunoreactive materials appeared qualitatively similar, and quantitative image analysis showed similar gradients of spectrin and Na+,K+-ATPase immunofluorescence along the long axis of elongated photoreceptors. Moreover, detergent extractability of alpha spectrin and the ATPase showed similar modifications in response to changes in pH or KCl concentration. ATPase detergent-extractable and mobile fractions were not changed in cultures treated with cytoskeletal inhibitors such as nocodazole. These data are consistent with a role for an asymmetrically distributed, spectrin-containing subcortical cytoskeleton in the preferential accumulation of Na+,K+-ATPase in the photoreceptor inner segment.