The Na(+)-K(+)-ATPase presents several different isoforms of its alpha- and beta-subunits. We detected alpha 1- and beta 1-mRNA transcripts and polypeptides in 3T3-L1 fibroblasts; during differentiation into adipocytes, alpha 1-mRNA decreased, alpha 2-mRNA was induced, beta 1-mRNA dropped to undetectable levels, and beta 2-mRNA was never expressed, suggesting that 3T3-L1 adipocytes may express an unidentified Na(+)-K(+)-ATPase beta-subunit isoform. Insulin rapidly increased ion pump activity [ouabain-sensitive 86Rb+(K+) uptake] in 3T3-L1 fibroblasts and adipocytes without changing the plasma membrane concentration of alpha 1- or alpha 2-subunits as determined by subcellular membrane fractionation and immunoblotting or by [3H]ouabain binding to intact cells. Monensin, which raises the concentration of intracellular Na+, increased Na(+)-K+ pump activity, and no further stimulation was achieved with insulin. The stimulation of the pump by insulin was reduced by bumetanide, an inhibitor of the Na(+)-K(+)-2Cl- cotransporter, and was prevented by omission of extracellular Cl-. Insulin increased both ouabain-sensitive and bumetanide-sensitive 86Rb+(K+) uptake. These results suggest that insulin activation of the Na(+)-K(+)-ATPase in 3T3-L1 adipocytes is mediated by an elevation in intracellular Na+ that is likely the consequence of Na(+)-K(+)-2Cl- cotransporter activation.