We have constructed and characterized transgenic Drosophila lines with modified Na(+),K(+)-ATPase activity. Using a temperature dependent promoter from the hsp70 gene to drive expression of wild-type alpha subunit cDNA, we can conditionally rescue bang-sensitive paralysis and ouabain sensitivity of a Drosophila Na(+),K(+)-ATPase alpha subunit hypomorphic mutant, 2206. In contrast, a mutant alpha subunit (alpha(D369N)) leads to increased bang-sensitive paralysis and ouabain sensitivity. We can also generate temperature dependent phenotypes in wild-type Drosophila using the same hsp70 controlled alpha transgenes. Ouabain sensitivity was as expected, however, both bang sensitive paralysis or locomotor phenotypes became more severe regardless of the type of alpha subunit transgene. Using the Gal4-UAS system we have limited expression of alpha transgenes to cell types that normally express a particular Drosophila Na(+),K(+)-ATPase beta (Nervana) subunit isoform (Nrv1 or 2). The Nrv1-Gal4 driver results in lethality while the Nrv2-Gal4 driver shows reduced viability, locomotor function and uncontrolled wing beating. These transgenic lines will be useful for disrupting function in a broad range of cell types.