An ultramicro method for the determination of glutaminase activity is described. Glutaminases I (phosphate activated) and II (pyruvate activated) and non activated glutaminase has been determined in fresh homogenate from 10 mg of tissue. The optimum conditions for glutaminase assay have been investigated in the kidney of man, dog and rat. In human kidney taken at autopsy no glutaminase I activity was found ; whereas in fresh human kidney biopsy specimens the glutaminase activity was of the same order of magnitude as in the dog and rat kidney. The variation over three orders of magnitude of glutaminase activity reported in the literature for each species was interpreted as the result of the instability of the enzymes, differences in assay conditions and dependence of glutaminase activity on the age of the animal.