The genetic environment of the blaOXA-18 geneencoding a peculiar clavulanic acid-inhibited Ambler class D extended-spectrum beta-lactamase was determined from the prototype OXA-18-producing Pseudomonas aeruginosa MUS clinical isolate. An 8.2-kb genomic DNA fragment containing blaOXA-18 gene was cloned from P. aeruginosa MUS. While most oxacillinases are integron-located, blaOXA-18 lacked gene cassette specific features. It was bracketed by two duplicated sequences, containing ISCR19, a novel insertion sequence of the ISCR family of mobile elements, DeltaintI1, a truncated integrase gene and, a truncated Deltaaac6' Ib gene cassette. It is likely that ISCR19 was at the origin of the blaOXA-18 gene mobilization by a Rolling Circle (RC) transposition event followed by homologous recombination. Furthermore, analysis of the cloned genomic DNA fragment revealed the presence of the integron-containing blaOXA-20 gene. Concommitantly, three P. aeruginosa clinical isolates, displaying a synergy image by double-disk diffusion tests on cloxacillin-containing plates, were isolated from three patients hospitalized in different wards over a nine-month period at the Saint-Luc University hospital (Brussels, Belgium). These isolates were positive by PCR for blaOXA-18 andblaOXA-20 genes, genetically related to P. aeruginosa MUS by PFGE and carried the same blaOXA-18/blaOXA-20 associated genetic structures. This report characterized the genetic elements likely at the origin of blaOXA-18 gene mobilization in P. aeruginosa and suggests the spread of this type of OXA-ESBL in P. aeruginosa in the Saint-Luc University hospital of Brussels, Belgium.