A coryneform bacterium that is able to utilize cis- and trans-3-chloroacrylic acid as sole carbon source for growth was isolated from freshwater sediment. The organism was found to produce two inducible dehalogenases, one specific for the cis- and the other for trans-3-chloroacrylic acid. Both dehalogenases were purified to homogeneity from cells induced for dehalogenase synthesis with 3-chlorocrotonic acid. The enzymes produced muconic acid semialdehyde (3-oxopropionic acid) from their respective 3-chloroacrylic acid substrate. No other substrates were found. The cis-3-chloroacrylic acid dehalogenase consisted of two polypeptide chains of a molecular weight 16.2 kDa. Trans-3-chloroacrylic acid dehalogenase was a protein with subunits of 7.4 and 8.7 kDa. The subunit and amino acid compositions and the N-terminal amino acid sequences of the enzymes indicate that they are not closely related.