We have cloned DNA fragments of plasmid pFL40 fromAlcaligenesxylosoxidans ssp. denitrificans ABIV encoding a D,L-2-haloalkanoic acid halidohydrolase (DhlIV). A 6.5-kb EcoRI/SalI-fragment with inducible expression of the halidohydrolase was cloned in Pseudomonas fluorescens and Escherichia coll. A 1.9-kb HindlI-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coll. The nucleotide sequence of this DNA-fragment was determined. It had an open reading frame coding for 296 amino acid residues (molecular weight of 32783 D). The dhllV gene showed sequence homology to a short segment of a D-specific dehalogenase (hadD) from Pseudomonasputida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhllV and the D,L-isomer specific dehI dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a ty54-dependent activator protein, similar to the dehRs regulatory gene from Pseudomonas putida PP3 is located upstream of dhllV. In contrast to DehI, dehalogenation of D-or L-chloropropionic acid by the DhllV-protein leads to lactic acid of inverted configuration.