Chemically induced hypoxia has been shown to induce a depletion of ATP. Since intracellular free Mg2⁢ ([Mg2⁢]i) appears to be tightly regulated following cellular energy depletion, we hypothesized that the increase in [Mg2⁢]i would result in Mg2⁢ extrusion following hormonal stimulation. To determine the relation between Mg2⁢ efflux and cellular energy state in a hypoxic rat heart and isolated myocytes, [Mg2⁢]i, ATP and Mg2⁢ content were measured by using mag-fura-2, luciferin-luciferase and atomic absorbance spectrophotometry. Mg2⁢ effluxes were stimulated by norepinephrine (NE) or cAMP analogues, respectively. Mg2⁢ effluxes induced by NE or cAMP were more stimulated in the presence of metabolic inhibitors (MI). Chemical hypoxia with NaCN (2 mM) caused a rapid decrease of cellular ATP within 1 min. Measurement of [Mg2⁢]i confirmed that ATP depletion was accompanied by an increase in [Mg2⁢]i. No change in Mg2⁢ efflux was observed when cells were incubated with MI. In the presence of MI, the cAMP-induced Mg2⁢ effluxes were inhibited by quinidine, imipramine, and removal of extracellular Na⁢. In addition, after several min of perfusion with Na⁢-free buffer, a large increase in Mg2⁢ efflux occurred when Na⁢-free buffer was switched to 120 mM Na⁢ containing buffer. A similar Mg2⁢ efflux was observed in myocytes. These effluxes were inhibited by quinidine and imipramine. These results indicate that the activation of Mg2⁢ effluxes by hormonal stimulation is directly dependent on intracellular Mg2⁢ contents and that these Mg2⁢ effluxes appear to occur through the Na⁢-dependent Na⁢/Mg2⁢ exchange system during chemical hypoxia.