The presence of multiple isoforms of the α and β subunits of the Na, K-ATPase in most mammalian tissues has hindered the understanding of the roles of the individual isoforms in directing Na, K-ATPase function. Expression of the Na, K-ATPase subunits in insect cells using recombinant baculoviruses has proven to be a useful system for the study of Na, K-ATPase function. Using this system, we have expressed the rat Na, K-ATPase α2β1 and α2β2 isoforms in Sf-9 insect cells, a cell line derived from the ovary of the fall armyworm, Spodoptera frugiperda. Both α1 and β2 isoforms can efficiently assemble with the α2 subunit to produce catalytically competent Na, K-ATPase molecules. The analysis of the kinetic properties of both isozymes showed that α2β1 and α2β2 have equivalent sensitivities to ouabain, and similar turnover numbers and apparent affinities for K+ and ATP. The dependence on Na+, however, differs between the isozymes, with α2β2 displaying a slightly higher apparent affinity for the cation than α2β1. In addition, the even greater kinetic differences between Na, K-ATPase isozymes varying in α isoforms may be important in further differentiating the enzyme. Thus, when compared to the rat α1β1 Na, K-ATPase expressed in Sf-9 cells, the α2β1 and α2β2 isozymes have a lower apparent affinity for K+ and a higher affinity for Na+ and ATP. Moreover, the α1β1 isozyme is approximately 250 times more resistant to ouabain than α2β1 and α2β2. These different kinetic characteristics of the Na, K-ATPase isozymes may help establish the ionic milieu required by tissues to meet their specific physiological requirements.