Two molecular forms of Na,K-ATPase can be isolated from the central nervous system. The two forms can be distinguished by their sensitivities to cardiac glycosides and by the electrophoretic mobilities of their catalytic subunits, a and a(+). Because Na,K-ATPase is a membranebound enzyme, it would be predicted to move in the rapid phase of axonal transport, and this was used as a means to determine which form(s) is made by a defined neuron of the central nervous system. Retinal ganpglion cells were labeled in vivo by intravitreal injection of [3 Simethionine; the Na,KATPase that was axonally transported down the optic nerve was purified, and the a and a(+) forms were separated by electrophoresis and detected by fluorography. The two forms were synthesized in the retina in approximately equal amounts. The a(+) form was the predominant form transported from the retinal ganglion cells to the lateral geniculate nucleus and superior colliculus. The oligodendrocytes and other sheath cells of the excised optic nerve, in contrast, synthesized only the a form when incubated in vitro with [35S]methionine. The labeled Na,K-ATPase found at the nerve endings always included a small amount of the a form in addition to the a(+) form. The proportions of the two forms did not change with time after transport, and the presence of labeled a was not affected by infusion of cycloheximide to inhibit intracranial protein synthesis. Hence, although a(+) is the predominant form, the evidence suggests that small amounts of the a form are also made and transported by retinal ganglion cells.The ouabain-inhibited Na,K-ATPase is the enzyme responsible for active transport of Na' and K+ across the cell membrane (1), and is present in both neurons and glia. When isolated from the kidney, it is comprised of a catalytic subunit of Mr approximately 95,000 (a) and a smaller glycoprotein subunit (13) of unknown function. Preparations of Na,KATPase from the brain, however, have two biochemically distinct forms of the catalytic subunit, called a and a(+) (2). These differ in electrophoretic mobility in NaDodSO4-containing gels (2-4) and in sensitivity to cardiac glycosides (2, 5, 6). Purified axolemmal membrane from rat brain white matter contains only a(+) (2, 3), while cultured glia contain only a (2), which suggests that a(+) is characteristic of neurons and a, of nonneuronal cells. Neurons of the sympathetic nervous system contain only the a form, however, and synaptosomes from the cerebral cortex contain both forms (2). Although the presence of the a form in synaptosomes may be due to contamination with membrane of glial origin, the evidence is consistent with the hypothesis that neurons can express either form or both. A selective labeling technique is needed to determine which forms are expressed in different neurons of the central nervous system. The Na,K-ATPase of a defined population of neurons was examined by taking advantage of the axonal transport of newly synthesized radioactively labeled protein to sites distant from the cell b...