Yeast artificial chromosomes (YACs) can accommodate large inserts and hence should be attractive tools for intra- and interspecies comparisons of bacterial genomes. YAC libraries were constructed from size-selected partial digests of human and Pseudomonas aeruginosa PAO DNA and SpeI-restricted PAO DNA. Whereas YACs from human DNA had an average size of 350 kilobase pairs (kbp), a P. aeruginosa sequence larger than 120 kbp was absent or truncated in the eukaryotic host. Coligation occurred for YACs smaller than 40 kbp, but stable YACs with 40-120 kbp large inserts of P. aeruginosa DNA were obtained in high yield. SpeI-restricted chromosomes from 97 P. aeruginosa strains representing 47 genotypes were hybridized with stable YACs from three equidistant regions of the PAO genome. The low complexity of hybridizing bands demonstrated that the analyzed 100 kbp sequence contigs were stably maintained in most P. aeruginosa isolates from both disease and environmental habitats.