Although the enzyme (Na + + K +)-ATPase has been extensively characterized, few studies of its major role, ATP-dependent Na + pumping, have been reported in vesicular preparations. This is because it is extremely difficult to determine fluxes of isotopic Na + accurately in most isolated membrane systems. Using highly purified cardiac sarcolemmal vesicles, we have developed a new technique to detect relative rates of ATP-dependent Na + transport sensitively. This technique relies on the presence of Na +-Ca 2+ exchange and ATP-driven Na + pump activities on the same inside-out sarcolemmal vesicles. ATP-dependent Na + uptake is monitored by a subsequent Na i +-dependent Ca 2+ uptake reaction (Na +-Ca 2+ exchange) using 45Ca 2+. We present evidence that the Na +-Ca 2+ exchange will be linearly related to the prior active Na + uptake. Although this method is indirect, it is much more sensitive than a direct approach using Na + isotopes. Applying this method, we measure cardiac ATP-dependent Na + transport and (Na + + K +)-ATPase activities in identical ionic media. We find that the (Na + + K +)-ATPase and the Na + pump have identical dependencies on both Na + and ATP. The dependence on [Na +] is sigmoidal, with a Hill coefficient of 2.8. Na + pumping is half-maximal at [Na +] = 9 mM. The K m for ATP is 0.21 mM. ADP competitively inhibits ATP-dependent Na + pumping. This approach should allow other new investigations on on ATP-dependent Na + transport across cardiac sarcolemma.