Protein mass spectrometry and molecular cloning techniques were used to identify and characterize mobile o-halobenzoate oxygenase genes in Pseudomonas aeruginosa strain JB2 and Pseudomonas huttiensis strain D1. Proteins induced in strains JB2 and D1 by growth on 2-chlorobenzoate (2-CBa) were extracted from sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and analyzed by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Two bands gave significant matches to OhbB and OhbA, which have been reported to be the  and  subunits, respectively, of an ortho-1,2-halobenzoate dioxygenase of P. aeruginosa strain 142 (T. V. Tsoi, E. G. Plotnikova, J. R. Cole, W. F. Guerin, M. Bagdasarian, and J. M. Tiedje, Appl. Environ. Microbiol. 65:2151-2162, 1999). PCR and Southern hybridization experiments confirmed that ohbAB were present in strain JB2 and were transferred from strain JB2 to strain D1. While the sequences of ohbA from strains JB2, D1, and 142 were identical, the sequences of ohbB from strains JB2 and D1 were identical to each other but differed slightly from that of strain 142. PCR analyses and Southern hybridization analyses indicated that ohbAB were conserved in strains JB2 and D1 and in strain 142 but that the regions adjoining these genes were divergent. Expression of ohbAB in Escherichia coli resulted in conversion of o-chlorobenzoates to the corresponding (chloro)catechols with the following apparent affinity: 2-CBa  2,5-dichlorobenzoate > 2,3,5-trichlorobenzoate > 2,4-dichlorobenzoate. The activity of OhbAB_JB2 appeared to differ from that reported for OhbAB₁₄₂ primarily in that a chlorine in the para position posed a greater impediment to catalysis with the former. Hybridization analysis of spontaneous 2-CBa mutants of strains JB2 and D1 verified that ohbAB were lost along with the genes, suggesting that all of the genes may be contained in the same mobile element. Strains JB2 and 142 originated from California and Russia, respectively. Thus, ohbAB and/or the mobile element on which they are carried may have a global distribution.