The coexpression of multiple isoforms of the alpha and beta subunits of the Na,K-ATPase in mammalian tissues gives rise to the complex molecular heterogeneity that characterizes the Na pump. The expression of the different Na,K-ATPase isoforms in insect cells using recombinant baculoviruses represents a useful system for the analysis of Na,K-ATPase isoform function. In the present study, we use this system to direct the expression of the rat Na,K-ATPase alpha 3 beta 1 and alpha 3 beta 2 in sf-9 cells, a cell line derived from the ovary of the fall armyworm, Spodoptera frugiperda. The association of alpha 3 with either beta 1 or beta 2 results in catalytically competent Na,K-ATPase isozymes. Analysis of the kinetic characteristics of these enzymes demonstrates that the accompanying beta subunit isoform does not drastically affect the properties of the alpha 3 polypeptide. This is evidenced by the similar turnover numbers, apparent affinities for K+ and ATP, and the comparable high sensitivity to ouabain exhibited by both isozymes. The kinetic dependence on Na+, however, is different for both isozymes, with alpha 3 beta 2 displaying a 1.6-fold higher apparent affinity for the cation than alpha 3 beta 1. Comparison with other Na,K-ATPase isozymes shows that the apparent Na+ affinity of alpha 3 beta 2 is similar to that of the alpha 1 beta 1 Na pump widely expressed in every tissue; nevertheless, its reactivity toward K+, ATP, and ouabain are characteristic of the alpha 3 isoform. The most pronounced kinetic differences in Na,K-ATPase function are a result of variations in alpha isoform composition.(ABSTRACT TRUNCATED AT 250 WORDS)