The genes encoding aniline utilization in Pseudomonas sp. strain CITI have been cloned in Escherichia coli and partially characterized. Molecular cloning of the genes was achieved by construction of a cosmid library, followed by mobilization of the library into mutants of Ps. sp. CITI impaired in a number of functions necessary for growth on aniline. A 42-kb Sau3A fragment was found to encode the ability to utilize aniline and contained the catechol 2,3-dioxygenase (C230) gene. The regions encoding these activities were subcloned and further characterized. Plasmids containing the aniline oxidase gene encoded a 260-kDa protein complex, which was putatively shown to be composed of 72 kDa and possibly 36 kDa subunits. The fragment required for C230 activity encodes a 35 kDa protein, similar in size to C230 gene products previously characterized.