Different isoforms of the sodium/potassium adenosinetriphosphatase (Na,K-ATPase) α and β subunits have been identified in mammals. The association of the various α and β polypeptides results in distinct Na,K- ATPase isozymes with unique enzymatic properties. We studied the function of the Na,K-ATPase α4 isoform in Sf-9 cells using recombinant baculoviruses. When α4 and the Na pump β1 subunit are coexpressed in the cells, Na,K- ATPase activity is induced. This activity is reflected by a ouabain-sensitive hydrolysis of ATP, by a Na+-dependent, K+-sensitive, and ouabain- inhibitable phosphorylation from ATP, and by the ouabain-inhibitable transport of K+. Furthermore, the activity of α4 is inhibited by the P-type ATPase blocker vanadate but not by compounds that inhibit the sarcoplasmic reticulum Ca-ATPase or the gastric H,K-ATPase. The Na,K-ATPase α4 isoform is specifically expressed in the testis of the rat. The gonad also expresses the β1 and β3 subunits. In insect cells, the α4 polypeptide is able to form active complexes with either of these subunits. Characterization of the enzymatic properties of the α4β1 and α4β3 isozymes indicates that both Na,K-ATPases have similar kinetics to Na+, K+, ATP, and ouabain. The enzymatic properties of α4β1 and α4β3 are, however, distinct from the other Na pump isozymes. A Na,K-ATPase activity with similar properties as the α4-containing enzymes was found in rat testis. This Na,K-ATPase activity represents approximately 55% of the total enzyme of the gonad. These results show that the α4 polypeptide is a functional isoform of the Na,K-ATPase both in vitro and in the native tissue.