A novel method for random mutagenesis of targeted chromosomal regions in Pseudomona aeruginosa was developed. This method can be used with a cloned DNA fragment of indefinite size that contains a putative gene of interest. Cloned DNA is digested to produce small fragments that are then randomly reassembled into long DNA inserts by using cosmid vectors and λ packaging reaction. This DNA is then transferred into P. aeruginosa and forced into the chromosome via homologous recombination, producing in a single step a random set of insertional mutants along a desired region of the chromosome. Application of this method to extend the analysis of the alginate regulatory region, using a cloned 6.2-kb fragment with the algr gene and the previously uncharacterized flanking regions, produced several insertional mutations. One mutation was obtained in algR, a known transcriptional regulator of mucoidy in P. aeruginosa. The null mutation of algR was generated in a mucoid derivative of the standard genetic strain PAO responsive to different environmental factors. This mutation was used to demonstrate that the algR gene product was not essential for the regulation of its promoters. Additional insertions were obtained in regions downstream and upstream of algR. A mutation that did not affect mucoidy was generated in a gene located 1 kb upstream of algR. This gene was transcribed in the direction opposite that of algR transcription and encoded a polypeptide of 47 kDa. Partial nucleotide sequence analysis revealed strong homology of its predicted gene product with the human and yeast argininosuccinate lyases. An insertion downstream of algR produced a strain showing reduced induction of mucoidy in response to growth on nitrate as the nitrogen source.