Studies in man and other mammals have demonstrated the existence of two forms of arginase, a cytoplasmic form located primarily in liver and a mitochondrial form expressed in lesser amounts in a larger number of organs, but especially kidney. They appear to be encoded in different gene loci. Using a colloidal silica gradient separation technique, we have now located arginase in H4 cells, a rat hepatoma-derived line, to the cytoplasm and the arginase in human embryonic kidney-derived line, to the mitochondrion. Antibody prepared against A1 precipitates all the arginase from liver, 50% from kidney and none of the activity from human embryonic kidney (HEK) cells. An antibody prepared against partially purified All, by contrast, precipitates > 90% of arginase activity from HEK cells, half from kidney and virtually none from H4 cells or rat liver.