When 3T3 preadipocyte cell lines are induced to differentiate to the adipocyte phenotype, the expression of Na(+)-K(+)-ATPase isoforms changes. Some disparities have been noted by investigators who used different hormonal conditions to stimulate adipocyte conversion, however. In the present report we investigated the effect of high concentrations of insulin on the 3T3-L1 cell line, to determine whether it affected Na(+)-K(+)-ATPase expression separately from its ability to promote phenotypic conversion. The effect of insulin was compared with that of dexamethasone and 3-isobutyl-1-methylxanthine. Changes in Na(+)-K(+)-ATPase subunit expression were seen regardless of the hormonal stimulus. Induction of alpha 2-mRNA and reduction of beta 1-mRNA were always observed. At the protein level, too, induction of alpha 2-protein was noted; alpha 1-protein levels were not markedly affected. The change in alpha-isoform protein and mRNA levels did not correspond quantitatively with the fraction of cells that were morphologically converted, suggesting that it is an early event in differentiation.