The three fractions of acyl-CoA synthetase differing in isoelectric pH were isolated from Escherichia coli by isoelectric focusing and characterized. They had the same molecular weight and identical immunochemical properties. The three fractions differed appreciably in pH-velocity profiles. These fractions had distinguishable thermal stabilities and peptide patterns obtained after limited proteolysis. Apparent Km and Vmax values for fatty acids were also significantly different in these fractions, although the specificity ranged from C8 to C18 fatty acids with maximum activity for lauric acid in all fractions.