4-Chlorobenzoyl coenzyme A dehalogenase was purified to homogeneity from Arthrobacter sp. strain 4-CB1 (previously known as Acinetobacter sp. strain 4-CB1), a bacterium isolated from PCB-contaminated soil. Purification was accomplished by four chromatographic steps, including a novel affinity chromatography step. 4-Chlorobenzoyl CoA dehalogenase is a homotetramer of 33-kDa subunits with an isoelectric point of 6.1. The enzyme is stable between pH 6.5 and 10. The optimum pH for kcat is pH 8. The enzyme is able to dehalogenate substrates bearing fluorine, chlorine, bromine and iodine in the 4-position, although the rate of dehalogenation of 4-fluorobenzoyl CoA is quite slow. The enzyme is specific for dehalogenation at the 4-position, as 3-chloro- and 2-chlorobenzoyl CoA are not dehalogenated. The N-terminal sequence of the Arthrobacter sp. strain 4-CB1 dehalogenase is almost identical to that of the 4-chlorobenzoyl CoA dehalogenase from Arthrobacter sp. strain SU and shows 30% identity to that from Pseudomonas sp. strain CBS-3.