Lung Na-K-ATPase has been shown to contribute to vectorial Na+ transport and edema clearance. The alpha 1- and beta 1-Na-K-ATPase subunits have been localized to alveolar type II (ATII) cells, and the alpha 2-Na-K-ATPase has been reported in rat lung homogenates. Expression of Na-K-ATPase alpha 1-, alpha 2-, and beta 1-subunits was investigated in rat ATII cells cultured for 7 days, a period during which they lose their phenotypic markers and differentiate to an alveolar type I (ATI)-like cell phenotype. Differentiation of ATII cells to an ATI-like phenotype resulted in a decrease of alpha 1- and an increase of alpha 2-mRNA and protein abundance without changes in the beta 1-subunit. Thus ATI-like cells exhibited a mixture of alpha 1- and alpha 2-isoforms. Nuclear run-on analysis suggests that these changes were transcriptionally regulated. The existence of the distinct functional classes of Na-K-ATPase in ATII and ATI-like cells was confirmed by ouabain inhibition of Na-K-ATPase activity. Ouabain inhibition of ATII cells was consistent with expression of the alpha 1-isozyme [50% inhibitory concentration (IC50) = 4 x 10(-5) M], whereas, in ATI-like cells, it was consistent with the presence of both alpha 1- and alpha 2-isozymes (IC50 = 9.0 x 10(-5) and 1.5 x 10(-7) M, respectively); [3H]ouabain binding studies corroborated these findings. Our results indicate that, during ATII cell cytodifferentiation with time in culture, there is a shift in isoform composition that may reflect physiological functions of alveolar epithelial cells.