The aim of the present work was to study the occurrence, distribution and diversity of 1,2-dichlorocatechol dioxygenase genes among 2,4-dichlorophenoxyacetic acid degrading bacteria. Phylogenetic relationships between the 31 strains or isolates were evaluated by amplified ribosomal DNA restriction analysis of the 16S rDNA gene. All the strains could be assigned to the β or γ subdivisions of the Proteobacteria. tfdC genes were detected by PCR amplification using degenerated primers. Two specific probes were produced from Ralstonia eutropha strain JMP134 and from a soil isolate strain PLAE6 which was grouped with Variovorax paradoxus. Sequence analysis of the probes revealed that they were homologous to the tfdC genes of JMP134 located on plasmid pJP4 and to the tfdC gene of Pseudomonas putida strain PaW85 located on plasmid pEST4011. The localization and the copy number of tfdC genes were determined by hybridization of plasmid profiles and genomic DNA restriction fragment length polymorphism profiles with the two probes. Most of the strains were found to bear tfdC genes on plasmids ranging from 78 to 532 kb; two strains without any plasmids were also found to hybridize with the probes, revealing a chromosomal localization of catabolic genes. Sequence analysis of the PCR products from different strains confirmed that four different classes of chlorocatechol 1,2-dioxygenase genes were present in the strains and isolates studied.