Abstract: Cupriavidus necator JMP134 has been extensively studied because of its ability to degrade chloroaromatic compounds, including the herbicides 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid (3-CB), which is achieved through the pJP4-encoded chlorocatechol degradation gene clusters: tfdC(I)D(I)E(I)F(I), and tfdD(II)C(II)E(II)F(II). The present work describes a different tfd-genes expression profile depending on whether C. necator cells were induced with 2,4-D or 3-CB. By contrast, in vitro binding assays of the Purified transcriptional activator TfdR showed similar binding to both tfd intergenic regions; these results were confirmed by in Vivo Studies of the expression of transcriptional lacZ fusions for these intergenic regions. Experiments aimed at investigating whether other pJP4 plasmid or chromosomal regulatory proteins could contribute to the differences in the response of both tfd promoters to induction by 2,4-D and 3-CB showed that the transcriptional regulators from the benzoate degradation pathway, CatR I and CatR2, affected 3-CB- and 2,4-D-related growth capabilities. It was also determined that the ISJP4-interrupted protein TfdT decreased growth on 3-CB. In addition, an ORF with 34% amino acid identity to IcIR-type transcriptional regulator members and located near the tfd(II) gene cluster module was shown to modulate the 2,4-D growth capability. Taken together, these results Suggest that tfd transcriptional regulation in C. necator JMP134 is far more complex than previously thought and that it involves proteins from different transcriptional regulator families. [Int Microbiol 2009; 12(2):97-106]