1. In broken red cell membranes, Mg2+ inhibition of Na+,K+-adenosine-5'-triphosphatase (Na+,K+-ATPase) activity was partially competitive with MgATP. Mg2+ inhibition of Na+,K+-ATPase activity was uncompetitive with K+ in broken red cell membranes, and Mg2+ inhibition of ouabain-sensitive K+ influx in K+-free resealed ghosts was uncompetitive with external K+. 2. When Na+,K+-ATPase activity was measured at relatively high K+ concentration, Mg2+ inhibition was partially competitive with Na+. Mg2+ inhibition of ouabain-sensitive K+ influx in K+-free resealed ghosts was competitive with cell Na+. Magnesium was a more effective inhibitor of the uncoupled Na+ efflux in low-Na+ ghosts than in high-Na+ ghosts. 3. These findings indicate that Mg2+ inhibition results from combination of the ion with the enzyme form E2K at high intracellular Na+ and K+, and from combination with the form E1 at low intracellular Na+ and K+. 4. In ghosts containing high concentrations of MgPO4, inhibition of the K+-K+ exchange by Mg2+ was more effective at high than at low nucleotide concentrations. At high MgPO4 and low Mg2+ concentration the activity of the exchange increased monotonically with nucleotide concentration, but at a higher Mg2+ concentration, nucleotide activation of the exchange was biphasic: the K+-K+ exchange rate increased, reached a maximum, and then decreased with increasing nucleotide concentration.