Cerebellar Purkinje neurons of rats have been shown to exhibit a progressive increase in resting membrane potential as the animals develop postnatally. The magnitude of this increase was equivalent in magnitude to the increase in the depolarizing action of ouabain, consistent with a role for the Na(+)/K(+)-pump in the hyperpolarization. Ouabain binding sites in whole cerebellum also increased with age. The present study was undertaken to confirm that the increases in ouabain binding and the electrophysiological responses to ouabain were a consequence of increases in the sodium pump and to determine whether the changes seen at the whole organ level were reflective of changes taking place at the cellular level. Using antibodies directed against the alpha(1), alpha(2), and alpha(3) subunits of the Na(+)/K(+)-ATPase, rats between 13 and 19 days of age exhibited a statistically significant increase in the relative amount of the alpha(3) subunit at the level of the whole organ, as determined by Western and slot blot analyses, with no change in the levels of either the alpha(1) or the alpha(2) subunit. Using immunohistochemistry, the alpha(3) subunit was shown to increase in both the Purkinje cell layer and the white matter during this postnatal time period, while the alpha(1) subunit increased in the granular layer. These results support and extend previous work, which pointed to a role for the electrogenic sodium pump in the developmental increase in Purkinje cell membrane potential. Furthermore, the data provide a cellular mechanism underlying the increase in resting membrane potential, that is, by the specific modulation of the alpha(3) subunit isoform.