1. The objectives of this investigation were to determine the effect of inhibition of mitochondrial function on intracellular free Mg concentration, [Mg2+]i, in the heart and to determine whether the calcium channel antagonist nifedipine would alter the response. 2. Cardiac myocytes were prepared as primary cultures from 7-day-old chick embryonic hearts. [Mg2+]i was determined in single ventricular cells with mag-fura-2. 3. Inhibition of mitochondrial function with carbonyl cyanide m-chlorophenylhydrozone (CCCP, 3 microM, plus amobarbital, 3 mM, produced a cessation of cardiac contractile frequency that was reversible. This was associated with an increase in [Mg2+]i from 0.48 to 0.98 mM which returned to near basal levels with removal of the drugs. [Ca2+]i oscillations with cell contraction were diminished in the presence of CCCP plus amobarbital and returned to normal following their removal. 4. In contrast, CCCP plus iodoacetate led to increased [Mg2+]i beyond 2 mM which was associated with elevated [Ca2+]i and cell death. 5. Nifedipine did not alter the cardiac contractile response to CCCP plus amobarbital. The increment in [Mg2+]i produced by CCCP plus amobarbital was not altered by nifedipine. These data suggest that [Mg2+]i is regulated by mitochondrial metabolism and nifedipine does not alter the increase in [Mg2+]i produced by inhibition of mitochondrial function suggesting increments in [Mg2+]i were from internal sources. Nifedipine may not interfere with the potentially beneficial actions of increased [Mg2+]i.