Glutaminase activity was assayed in homogenates and mitochondria of kidneys from normal and acidotic rats. These preparations were also subjected to ultrasonic disintegration and the enzyme was assayed in the pellets and supernatants resulting from ultracentrifugation. Glutaminase activity was recovered mainly in the mitochondrial fraction of unsonicated tissue. Sonication released some of the glutaminase from the mitochondria. The increase in glutaminase activity due to the addition of phosphate was greater for the enzyme released from the mitochondria by sonication than for the enzyme recovered in the mitochondrial pellet after centrifugation. Acidosis did not significantly alter glutaminase activity when assayed in Tris buffer. However, when phosphate was present in the incubation medium acidosis increased glutaminase activity whether or not it remained attached to the mitochondrial membrane during sonication. The data indicates that there is more than one isoenzyme of glutaminase in kidney mitochondria and that the sensitivities of these isoenzymes to phosphate are not identical.