1. Rat heart homogenates catabolized glutamine in the presence of rotenone, an inhibitor of the respiratory chain. 2. The reaction was markedly stimulated by phosphate and inhibited by glutamate, but not greatly affected by ammonia. 3. Glutamine breakdown was enhanced by lactate, malate, citrate and ATP, and almost completely blocked by 1 mM-N-ethylmaleimide. 4. The Km for glutamine measured in homogenates supplemented with either 50 mM- or 100 mM-phosphate and at pH 7.3 or 8.0 was about 4 mM, whereas the Vmax was larger at higher anion concentrations and at the more alkaline pH. 5. Activity of glutaminase was 3-fold greater in isolated mitochondria than in muscle homogenates. Distribution of the activity was the same as that of a mitochondrial marker, cytochrome c oxidase. 6. Properties of glutaminase with respect to its dependence on the concentrations of phosphate, glutamine and H+ were the same in intact and broken mitochondria and very similar to those in homogenates. However, the specific activity of the enzyme was considerably smaller in frozen-thawed than in intact organelles. 7. It is concluded that heart mitochondria possess a kidney-type phosphate-activated glutaminase that can serve as a source of myocardial glutamate.