The occurrence of a new bacterial dehalogenase acting on both the optical isomers of 2-halogenated alkanoic acids was demonstrated. When the haloalkanoic acid-utilizing bacteria were screened in a medium containing DL-2-chloropropionate as a sole carbon source, two types of bacteria were isolated: (1) a few strains utilizing both D- and L-isomers of 2-chloropropionate and (2) strains utilizing only the L-isomer. A dehalogenating enzyme was obtained from the cells of Pseudomonas sp. which is able to utilize both isomers. The crude enzyme catalyzed the dehalogenation of D- and L-2-chloropropionates to yield L- and D-isomers of lactate, respectively. The enzyme showed the same pH optimum and heat inactivation rate for the D- and L-isomers. Apparent Km values for D- and L-2-chloropropionates were 4.5 and 1.0mM, respectively. The enzyme acted specifically on 2-haloalkanoic acids. Activity staining of disc-gels electrophoresed with the crude enzyme preparation showed that the dehalogenation of D- and L-2-chloropropionates, monochloroacetate, dichloroacetate, 2,2-dichloropropionate, and DL-2-chlorobutyrate is due to a single protein.