4-Chlorobenzoyl CoA dehalogenase catalyzes the replacement of the chlorine substituent on 4-chlorobenzoyl CoA with a hydroxyl group. The SNAr mechanism seems the most likely mechanism for this unusual and intrinsically difficult nucleophilic aromatic substitution reaction. However, the order of leaving group abilities observed for various 4-halobenzoyl CoA substrates is opposite that expected. Therefore, we have explored alternative mechanisms for the enzymic dehalogenation reaction. The aryne mechanism was ruled out by the absence of a deuterium kinetic isotope effect on the reaction. The S(RN)1 and S(ON)2 mechanisms were deemed unlikely because of the lack of evidence for a metal ion or organic cofactor on the enzyme. Thus, the dehalogenation reaction appears to occur via an SNAr mechanism, Further investigations suggested that the reaction proceeds by displacement of chloride by an enzymic carboxylate, followed by hydrolysis of an aryl-enzyme intermediate. When an alternative nucleophile, hydroxylamine, was included in reaction mixtures, no product derived from direct attack of hydroxylamine upon 4-chlorobenzoyl CoA could be detected. However, inclusion of higher concentrations of hydroxylamine (100 mM) resulted in inactivation of the enzyme. These data are consistent with the formation of an aryl-enzyme intermediate that is converted to a hydroxamic acid upon attack by hydroxylamine. Enzyme activity is recovered after hydroxylamine is removed, suggesting that the enzyme is able to slowly hydrolyze the hydroxamic acid and restore the active-site carboxylate, Single-turnover O-18-labeling experiments designed to confirm that the reaction occurs by direct attack of an active-site carboxylate to form an aryl-enzyme intermediate were difficult to interpret. Approximately one-half of the product contained oxygen derived from the solvent and one-half contained oxygen derived from the enzyme. Possible explanations for this phenomenon were explored, but a satisfactory explanation has not been found.