(A) Employing standard solutions, pump currents were initially activated twice by replacing 5 mM Na with 5 mM K. Removal of Na without application of K activates a small current, and the pump current activated subsequently with 5 mM K is less than one-half of the control pump current. Thus, pumps inactivate in nominally Na free solution, presumably because pump activity supported by contaminating K is adequate to allow inactivation. Pump currents recover in the presence of 5 mM Na, and the application of 15 mM has very little further rescue effect. From 6 experiments, employing 5, 15 and 120 mM Na, the half-maximal Na concentration required to rescue pump currents was 2.3 mM. (B) Under standard conditions, 120 mM Na was applied, instead of NMG, for 30 s before applying K in the absence of Na. Peak pump currents were increased by 13% on average, compared to currents activated after incubation with 7 mM Na. (B) Na/K pump inactivation is promoted by conditions that allow backward pump reactions from E2 to E1 states. With 10 mM deoxyglucose in the pipette solution, to promote ATP hydrolysis and generation of ADP by hexokinsaes, pump currents ran down slowly over several minutes. After application of 120 mM Na for 30 s, peak pump currents activated by K were decreased to nearly their 15 s value, as expected if inactivation had occurred in the presence of extracellular Na. This result verifies a key prediction of the inactivation model presented in Figure 1.