Three Klebsiella oxytoca isolates and one Klebsiella pneumoniae isolate from three children admitted to the Hematology Unit of Hospital Vall d'Hebron (Barcelona, Spain) exhibited a susceptibility pattern suggesting OXY β-lactamase hyperproduction. All the isolates contained a 95-kb plasmid that harbored blaOXY-1, which was transferred by electrotransformation but could not be self-transferred by conjugation. A qnrS1 gene was also harbored in the blaOXY-1-carrying plasmid. This is the first report of a plasmid-encoded OXY β-lactamase. Copyright © 2009, American Society for Microbiology. All Rights Reserved.