The subcellular distribution of adrenal glycerol kinase in man and rat are reported and the bisubstrate kinetics of the soluble enzyme are compared in these two species. The specific activity of glycerol kinase in human whole adrenal homogenate (145 microU/mg protein) was 3 times that found in rat whole adrenal homogenate (48 microU/mg protein). In both species 8% of the total glycerol kinase activity was associated with the nuclear pellet fraction. In human, 62% of the total activity was soluble, while 24% was associated with the postnuclear particulate fraction. Rat glycerol kinase activity was also predominantly soluble: 69% of the total activity was soluble and 13% was in the postnuclear particulate fraction. The apparent Km for glycerol in soluble adrenal glycerol kinase was similar in both species, 2.8 microM in human and 3.1 microM in rat. The apparent Km for ATP in soluble human adrenal glycerol kinase was 22.0 microM. In rat the enzyme did not appear to follow Michaelis-Menten kinetics with ATP as substrate. The Vmax for the soluble enzyme was similar in both human and rat. This report provides a background to biochemical investigations on human glycerol kinase deficiency, an inborn error of metabolism which may be characterized by adrenal hypoplasia and insufficiency.