A correlation was found between mitochondrial matrix free magnesium ([Mg2+]m), measured by means of the fluorescent indicator Mag-Fura-2, and citrulline synthesis in rat liver mitochondria. The variation of [Mg2+]m from 0.05 to 1.7 mM by changes in external Mg2+, induced a 20 +/- 8.5% increase in the rate of citrulline synthesis, whereas a further increase of [Mg2+]m to 3.3 mM induced a return to basal values. The increase in [Mg2+]m, as well as the diminution of external pH, also promoted an elevation of matrix free Ca2+ ([Ca2+]m). An increase in [Ca2+]m, at constant [Mg2+]m and pH, resulted in a 3-fold stimulation of citrulline synthesis. The data suggest that [Mg2+]m may modulate the rate of citrulline synthesis through a direct interaction with carbamoyl-phosphate synthase I (ammonia) and, indirectly, by changing the levels of matrix Ca2+.