The aniline-assimilating bacterium Acinetobacter lwoffii K24 has two cat gene clusters (cat(1) and cat(2)). In this study, we completely sequenced 10-kb DNA fragment of cat, genes of A. lwoffii K24, which had been cloned in plasmid pCD1-1. Sequence analysis revealed that the order of genes in the cat, operon-containing gene cluster was ORF porin, catR(1), catB(1)CA(1)D(1) ORF1, and ORF2. Two ORFs located immediately downstream catD were most similar with two ORFs in cat gene cluster of Acinetobacter calcoaceticus ADP1 but the gene structure of catR(1)B(1)C(1)A(1) was closest to that found in Frateurua sp. ANA-18 or Pseudomonas putida PRS2000. CatA(1) gene product was significantly overexpressed and detected in SDS-PAGE when four cat, genes (catB(1)C(1)A(1)D) were placed under the control of a lac promoter in pUC118 while overexpressions of other cat genes were accomplished under the control of a lac promoter in pET vector system. All gene products were verified by N-terminal amino acid sequencing. Gel retardation assay revealed that the putative regulatory gene activator CatR(1) for the catB(1)C(1)A(1) operon could bind the promoter region of catB(2) as well as catB(1), suggesting that transcription of catB(1), or catB(2) might be controlled by the putative gene activator CatR(1). However, the promoter regions of catA(1) and catA(2) were found to have no affinity with catR(1).