The subcellular distribution and substrate kinetics of soluble and particulate-associated bovine adrenal glycerol kinase have been investigated. Whole adrenal, adrenal cortex and adrenal medulla were examined for distribution of glycerol kinase between soluble and particulate fractions. No major differences in distribution were noted between these tissues; of the total homogenate activity, 0-20% sedimented with the nuclear fraction, 24 36% sedimented with the post-nuclear fraction and 64-69% remained soluble. Steadystate kinetic parameters of glycerol kinase activity were compared in the soluble and mitochondrial fractions. The K m for glycerol in the soluble fraction was 6.3 ยฑ 0.1 ~M and in the mitochondriM fraction was 4.0 ยฑ 0.3 #M. The K m for ATP in soluble fraction was 12.8 + 1.5 and in the mitochondrial fraction was 5.3 + 1.6. Release of adrenal glycerol kinase from the mitochondrial fraction was investigated using inorganic phosphate, ATP and glycerol 3-phosphate. Of these compounds, only ATP and glycerol 3-phosphate were effective in releasing particulate-associated glycerol kinase. Inorganic phosphate had no effect upon release. Particulate-associated glycerol kinase activity of the mitochondrial fraction was stimulated by addition of succinate and ADP and was inhibited by addition of atractyloside. The data presented here indicate that bound glycerol kinase found within the mitochondrial fraction is kinetically distinct from soluble glycerol kinase and binding to mitochondria is responsive to substrate and product levels within the physiological range.