The activity of rat liver glutaminase from sedimented fractions of freeze-thawed mitochondria is strongly affected by variation in the Mg2+ concentration within the approximate physiological range of activators. A rise in the Mg2+ concentration stimulates glutaminase by increasing the apparent affinity of the enzyme for its positive modifier phosphate. With the addition of 4 mM Mg2+ the M0.5 for phosphate activation decreased from 18 to 9.5 mM at pH 7.1, 10 to 5.8 mM at pH 7.4 and 6.4 to 4.0 mM at pH 7.7. The result is an increase in the apparent affinity of the enzyme for glutamine. With the addition of 4 mM Mg2+ the S0.5 of glutaminase for glutamine decreased from 24 to 13 mM at pH 7.1, 14 to 9.6 mM at pH 7.4, and remained unchanged at 8.2 mM at pH 7.7. Since Mg2+ stimulates glutaminase, as does a rise in pH (Szweda, L.I. and Atkinson, D.E. (1989) J. Biol. Chem. 264, 15357-15360), by increasing the apparent affinity of the enzyme for phosphate, it reduces the inhibitory effect of a decrease in pH and/or phosphate concentration over a physiologically relevant range.