A decrease in Na+/K+-pump activity is an early event of Friend murine erythroleukemic (MEL) cell differentiation along the erythroid pathway. This decreased Na+/K+-pump activity has been proposed to be an essential step in differentiation which would cause a rise in intracellular Na+ concentration and then, by means of Na+/Ca2+ exchange, an increase in intracellular Ca2+. An increase in intracellular Ca2+ has been proposed to be essential for induction of differentiation. A critical prediction of this Na+-Ca2+ hypothesis is the rise in intracellular Na+. To test this prediction we have measured intracellular Na+ using a novel triple isotope method involving 3H2O, [14C]sucrose, and 22Na to measure total water, extracellular fluid, and Na+, respectively. 22Na equilibration occurred in less than 10 min. In uninduced cells, intracellular Na+ was 15.2 +/- 2.2 mM (S.D., n = 22); after induction for 14-16 h with dimethyl sulfoxide, intracellular Na+ decreased significantly (p less than 0.0001) to 8.4 +/- 1.4 mM (n = 21). The time course of the decline in intracellular Na+ paralleled that of the decrease in the Na+/K+-pump activity. These results are in direct contradiction to the Na+-Ca2+ hypothesis and suggest that observed changes in Na+/K+-pump activity can be explained solely on the basis of changes in intracellular Na+. The drop in intracellular Na+ is due to a decrease in Na+ influx. We suggest, however, that the decrease in the Na+ influx is not itself an essential event of differentiation, but may be induced by a change in the flux of another ion coupled to Na+.