The objective of this study was to determine the prevalence of the plasmid-borne quinolone resistance genes qnrA, qnrB and qnrS in a collection of Enterobacteriaceae causing bacteraemia. The presence of the three genes was tested for using multiplex PCR in 306 clinical isolates. Plasmid analysis was performed using I-CeuI and S1 nuclease digestion and hybridization with specific probes for the qnr and 23S rRNA genes. Five strains were found to carry a qnr gene, one of which, qnrB16, a new variant of qnrB, was detected in a Citrobacter freundii isolate. The qnrB6 variant was found in two C. freundii isolates and in one Citrobacter werkmanii isolate. The qnrS2 gene was found in one Klebsiella pneumoniae isolate. The qnrA gene was not found in any of the isolates studied. The qnrS2 gene was located on a plasmid of c. 50 kb, whereas qnrB6 and qnrB16 were inserted in the chromosome between pspF and the orf2, which had previously been found in a complex integron. In the Hospital Clinic of Barcelona, Spain, the prevalence of qnrB was higher than that of qnrA and qnrS. The importance of the description of the new qnrB16 is emphasized.