1. To determine whether there is a change during differentiation, the activity and expression of Na(+)-K(+)-ATPase were studied in mononucleate cytotrophoblast cells (18 h culture) and syncytiotrophoblast-like cells (66 h culture). A choriocarcinoma-derived cell line (JAr) which, unlike the cytotrophoblast cells, divides in culture, was also studied for comparison. 2. Na(+)-K(+)-ATPase activity was assessed by measurement of ouabain-sensitive 86Rb+ uptake. Na(+)-K(+)-ATPase expression was determined by (i) measurement of [3H]ouabain binding and (ii) Northern hybridization to measure expression of alpha-1 and beta 1-subunit mRNA. 3. There was no significant difference in either activity or expression of Na(+)-K(+)-ATPase during differentiation of cytotrophoblast cells. However, expression of alpha 1- and beta 1-subunit mRNA was significantly lower in 66 vs. 18 h cultured cytotrophoblast cells. 4. Both Na(+)-K(+)-ATPase activity and [3H]ouabain binding was significantly greater in JAr cells than either cytotrophoblast cell groups, although expression of alpha 1- and beta 1-subunit mRNA was the same as cytotrophoblast cells cultured for 18 h. 5. It is concluded that N(+)-K(+)-ATPase activity and protein expression does not change during differentiation of cytotrophoblast cells but that there are changes in expression at the transcriptional or post-transcriptional level.