Pseudomonas putida Plll is able to utilize a broad range of monochlorinated, dichlorinated, and trichlorinated benzoates. The involvement of two separate dioxygenases was noted from data on plasmid profiles and DNA hybridization. The benzoate dioxygenase, which converts 3-chlorobenzoate (3-CB), 4-CB, and benzoate to the corresponding catechols via reduction of a dihydrodiol, was shown to be chromosomally coded. The chlorobenzoate-1,2-dioxygenase that converts ortho-chlorobenzoates to the corresponding catechols without the need of a functional dioldehydrogenase was shown to be encoded on plasmid pPBlll (75 kb). Cured strains were unable to utilize ortho-chlorobenzoates for growth. DNA hybridization data indicated that catabolism of the corresponding chlorocatechols was coded on chromosomal genes. Maintenance of plasmid pPBlll was dependent on the presence ofortho-chlorobenzoates in the growth media. A unique variant of Plll (PlIlD), able to grow on 3,5-dichlorobenzoate (3,5-DCB), was obtained by continuous subculturing from media containing progressively lower and higher concentrations of 3-CB and 3,5-DCB, respectively. The low frequency of segregants able to grow on 2,5-DCB, 2,3-DCB, and 2,3,5-trichlorobenzoate was evident by lag periods greater than 200 h. Continued subculture on 3,5-DCB resulted in the formation of new plasmid pPH111 (120 kb), which was homologous to pPBlll. A probe from the clc operon, which encodes for the chlorocatechol pathway, hybridized to plasmid pPH111 and to the chromosome of the wild-type strain Plll but not to its plasmid pPB111 nor to the chromosome of strain PlllA, which had lost the ability to utilize chlorobenzoates. These data indicate that the clc operon, which is located in the chromosome of wild-type strain Plll, is excised in variant PillA and translocated into plasmid pPH111 of variant P1liD.