Summary. The hydrolysis of glutamine by rat liver preparations occurred through a phosphate-dependent reaction differing from the one in rat kidney preparations primarily in its greater affinity for phosphate. The two enzymes also differed in pH optima, affinity for glutamine, reactions with various activators and inhibitors, and distributions in tissues. Both enzymes were mitochondrial, but retained their distinctive differences after sonication and in mixtures. The conditions for the independent assay of each type of activity in tissues are described. The liver type of reaction was limited to adult and regenerating liver, while high activities of the kidney-type of reaction were found in brain, kidney and small intestine and lesser activities in a variety of other tissues, including fetal liver and hepatoma. A third type of activity occurred independently of phosphate addition and comprised onesixth of the total in kidney preparations and less in other tissues.