Gene amplification in the chromosome of rec-2 Pseudomonas aeruginosa PAO2003 upon growth on kanamycin-supplemented media led to a stable mucoid phenotype. The chromosomal region controlling alginate biosynthesis was shown to be amplified four to six times as a direct tandem repeat of at least 16.8 kilobase pairs. This amplification was deduced from Southern DNA-DNA hybridization patterns of the chromosomal DNA digested with restriction endonucleases BglII and EcoRI and probed with a cloned DNA segment complementing the alg-22 mutation. The part of the amplified unit carrying the novel DNA joint was cloned. The EcoRI junction fragment was further subcloned and used to probe chromosomes of parental strain PAO2003 and mucoid variant VD2003M. As predicted, the EcoRI junction fragment hybridized to the two chromosomal fragments required to produce the novel junction. Though the mucoid phenotype caused by gene amplification was stable, nonmucoid revertants were obtained at a low frequency on tetracycline-containing media. Southern hybridization of chromosomal DNA from a nonmucoid revertant revealed a reduction in the copy number of amplified DNA. These results suggest a direct relationship between amplification of this chromosomal segment and the induction of mucoidy.