A mitochondrial freeze/thaw lysate was fractionated on a DEAE-cellulose column into four distinct acyl-CoA ligase fractions. First to elute was a 50 kDa short-chain ligase that activated only short-chain fatty acids. Next to elute were three ligases that had activity toward both medium-chain fatty acids and xenobiotic carboxylic acids; these were termed xenobiotic/medium-chain ligases (X-ligases) and labeled XL-I, XL-II, and XL-III, respectively, based on order of elution. The molecular weight of X-ligases I, II, and III were ca. 55,000, 55,500 and 53,000, respectively. Form XL-III showed no pH optimum; the rate increased steadily with pH beginning from pH 7.0. XL-I and XL-II showed the same behavior with benzoate as substrate, but with medium-chain fatty acids, both forms had a pH optimum at 8.8. The three X-ligases differed in substrate specificity. XL-I was the predominant nicotinic acid activating form and had the lowest Km for benzoate. Form XL-II was the only form with measurable salicylate activity, although it was extremely low. XL-III was the only 2,4,6,8-decatetraenoic acid activating form and also was the predominant medium-chain fatty acid-activating form. By comparison of substrate specificities, it was concluded that the two previously reported ligase preparations were mixtures of the three forms. When the ligase rates were compared to previously determined N-acyltransferase rates toward benzoyl-CoA and phenylacetyl-CoA, the data showed that ligase activities are 100-fold lower, and thus the ligase is rate limiting for the conjugation of both of these xenobiotics.