In 545 Pseudomonas aeruginosa strains, mainly collected from patients with cystic fibrosis, SpeI-DraI macrorestriction fragment length diversity was scanned for using probes of known map position on the P. aeruginosa PAO chromosome. Southern analysis of the 60 unrelated clones uncovered a gradient of macrorestriction fragment length polymorphisms (RFLPs) from the origin of replication towards the auxotroph-poor region of the P. aeruginosa genome. Linkage disequilibrium between macrorestriction sites was conserved in the P. aeruginosa population in the region encompassed by the rrn operons. The oriC-reactive SpeI fragment was conserved in nearly all isolates examined. Few fragment length classes were seen for the alg60-, algR- and toxA-encoding SpeI fragments. Fragment size varied within one class by up to 20 kb. Two probes from the auxotroph-poor region detected a broad size range for the SpeI fragment, suggesting extensive genomic diversity in these regions. Subclonal variation of fragment size was detected at all investigated loci in at least one of the analysed clones, but within one particular clone, SpeI-RFLPs were found at only few loci.