We have cloned DNA fragments of plasmid pFL40 from Alcaligenes xylosoxidans 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 coli. A 1.9-kb HindII-fragment demonstrated expression of the dehalogenase only due to the presence of the promoter from the pUC vector in Escherichia coli. 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 dhlIV gene showed sequence homology to a short segment of a D-specific dehalogenase (hadD) from Pseudomonas putida AJ1, but not to any other known DNA sequences. Restriction enzyme patterns indicated similarity between dhlIV and the D,L- isomer specific dehl dehalogenase gene from Pseudomonas putida PP3. There are some indications from restriction enzyme patterns and initial sequencing data, that a gene encoding a sigma 54-dependent activator protein, similar to the dehRI regulatory gene from Pseudomonas putida PP3 is located upstream of dhlIV. In contrast to DehI, dehalogenation of D- or L-chloropropionic acid by the DhlIV-protein leads to lactic acid of inverted configuration.