Agrobacterium tumefaciens RS5 harbors two different hydrolytic haloalkanoic acid dehalogenase genes, one coding for a nonstereospecific enzyme (DhlS5II) and a second for a cryptic L-isomer-specific dehalogenase (DhlS5I). The latter gene was cloned and expressed in Escherichia coli. Biochemical characterization and sequence analysis of dhlS5I shows its membership to the class of the L-isomer-specific hydrolytic dehalogenases. Highest homology of 72% was found to the dehalogenase LdexYL from Pseudomonas sp. YL. Both enzymes share an unusual high temperature optimum of 65 degrees C. Controlled by a vector promoter, high specific dehalogenase activities up to 32 U mg(-1) protein were obtained in E. coli, and putatively, owing to its own sigma(70)-dependent promoter, a constitutive low-level expression of dhlS5I of 0.4 U mg(-1) protein was measured.