Rat thymocytes showed two Na+/Mg2+ exchangers with high- and low- affinities for external Na+ (Na+o) at physiological internal Mg2+content. The total internal Mg2+ content (Mg2+it) was enhanced by loading with MgCl2 and the ionophore A-23187. Under these conditions, Na+/Mg2+ exchangers were dramatically stimulated by the Mg2+it increase. Na+-induced Mg2+ effluxes were independent of Cl-o or H+. The Na+/Mg2+ exchangers, which we named HANao (high affinity for Na+o) and LANao (low affinity for Na+o), were dissected in Mg2+-loaded thymocytes according to their kinetics and stoichiometries. HANao, which showed an apparent dissociation constant for Na+o (KNa H) = 9.2 +/- 1.6 mmol l(-1) Na+o and a maximal Na+ influx rate (VNa(Na H)max) = 30.5 +/- 6.1 mmol (l cells)(-1) h(-1), was a 1Na+:1Mg2+ simultaneous antiporter insensitive to external magnesium (Mg2+o) whereas that LANao, with KNa L = 65.1 +/- 8.6 mmol l(-1) Na+ and a VNa(Na L)max = 79.5 +/- 14.3 mmol (l cells)(-1) Na+ h(-1), was a 2Na+:1Mg2+ "ping-pong" antiporter which was strongly inhibited by Mg2+o. At physiological concentration of Mg2+o (1 mM), the Na+/Mg2+ exchange through the LANao was inhibited by approximately 50%. Amiloride (10(-4) M) inhibited at similar extent both Na+ and Mg2+ fluxes at high and at low Na+o.