Glycine conjugation is the most important route of detoxification, not only of many xenobiotic carboxylic acids but also of endogenous acids. However, the first reaction of glycine conjugation in some cases is reported to involve a class of active intermediates implicated in toxicity. The pathway of glycine conjugation consists of two sequential reactions. To elucidate the factors that regulate glycine conjugation, benzoic acid derivatives, acyl-CoAs as the active intermediates and glycine conjugates were simultaneously determined by LC-ESI-MS. Three compounds were collected from the microdialysis probe immersed into mitochondrial incubation mixture. A rate-limiting step was found to be the linkage to the amino group of glycine in a reaction that is catalyzed by acyl-CoA glycine: N-acyltransferases. However, the medium chain acyl-CoA synthetase catalyzing the initial reaction of glycine conjugation was the rate-limiting enzyme. Further, it was suggested that the stability of acyl-CoAs is one of the factors that determine the metabolic fate of xenobiotics containing a carboxylic group.