We have used the 2.0-kb DNA fragment Ω [Prentki and Krisch, Gene 29 (1984) 303–313] to mutagenize in vitro a broad-host-range plasmid carrying the entire meta-cleavage pathway of the Pseudomonas putida TOL plasmid pWW0. The mutant plasmids were subsequently introduced by conjugal mobilization into a variety of Gram-negative bacteria. The Ω fragment carries a selectable marker (aadA+; SpcR/SmR), which is expressed in all species tested, as well as flanking transcription and translation termination signals and synthetic Polylinkers. Expression of the plasmid-bome catechol 2,3-dioxygenase (C230) gene, situated downstream from the site of Ω insertion, was substantially reduced in all strains tested. The transcription terminators originally cloned from bacteriophage T4 gene 32, are apparently functional in a wide range of hosts. Insertional mutagenesis with the Ω ‘interposon' can thus be used in a wide variety of species, with the advantages of a positive selection for the presence of the fragment, the termination of RNA and protein synthesis beyond the site of insertion, and genetic stability of the resulting mutation.