This report describes the isolation and preliminary characterization of a 5.0-kilobase-pair (kbp) EcoRI DNA restriction fragment carrying the catBCDE genes from Acinetobacter calcoaceticus. The respective genes encode enzymes that catalyze four consecutive reactions in the catechol branch of the beta-ketoadipate pathway: catB, muconate lactonizing enzyme (EC 5.5.1.1); catC, muconolactone isomerase (EC 5.3.3.4); catD, beta-ketoadipate enol-lactone hydrolase (EC 3.1.1.24); and catE, beta-ketoadipate succinyl-coenzyme A transferase (EC 2.8.3.6). In A. calcoaceticus, pcaDE genes encode products with the same enzyme activities as those encoded by the respective catDE genes. In Pseudomonas putida, the requirements for both catDE and pcaDE genes are met by a single set of genes, designated pcaDE. A P. putida mutant with a dysfunctional pcaE gene was used to select a recombinant pKT230 plasmid carrying the 5.0-kbp EcoRI restriction fragment containing the A. calcoaceticus catE structural gene. The recombinant plasmid, pAN1, complemented P. putida mutants with lesions in catB, catC, pcaD, and pcaE genes; the complemented activities were expressed constitutively in the recombinant P. putida strains. After introduction into Escherichia coli, the pAN1 plasmid expressed the activities constitutively but at much lower levels that those found in the P. putida transformants or in fully induced cultures of A. calcoaceticus or P. putida. When placed under the control of a lac promoter on a recombinant pUC13 plasmid in E. coli, the A. calcoaceticus restriction fragment expressed catBCDE activities at levels severalfold higher than those found in fully induced cultures of A. calcoaceticus. Thus there is no translational barrier to expression of the A. calcoaceticus genes at high levels in E. coli. The genetic origin of the cloned catBCDE genes was demonstrated by the fact that the 5.0-kbp EcoRI restriction fragment hybridized with a corresponding fragment from wild-type A. calcoaceticus DNA. This fragment was missing in DNA from an A. calcoaceticus mutant in which the cat genes had been removed by deletion. The properties of the cloned fragment demonstrate physical linkage of the catBCDE genes and suggest that they are coordinately transcribed.