Mutant derivatives of a plasmid, pCF20, which carries the I-D fragment of the TOL plasmid pWW0 have been isolated using Tn5 transposon mutagenesis. Insertion mutations of the and regulatory genes of the catabolic pathway have been isolated and characterized and their ability to induce catechol 2,3-oxygenase activity determined. Analysis of the insertion mutants and also segments of the I-D fragment cloned into plasmid pUC8 in maxicells has identified a 68 kDa polypeptide product encoded by the gene. No clear candidate for the polypeptide was observed. The nucleotide sequence of the region, the intergenic region and part of the region has been determined and open reading frames (ORFs) assigned for both genes. The ORF designated appears capable of encoding a polypeptide of ~ 37 kDa.