Three epidemiologically and clonally unrelated Citrobacter werkmanii isolates of poultry origin from Germany were investigated for the mechanisms responsible for decreased fluoroquinolone susceptibility. The MICs, determined by broth macrodilution, varied between 8 and 256 g/ml for nalidixic acid and between 0.0625 and 1 g/ml for ciprofloxacin. The quinolone resistance-determining regions (QRDR) of gyrA, gyrB, parC, and parE were amplified and sequenced: CIT1 showed a Thr83Ile substitution in GyrA and a Ser57Thr substitution in ParC, CIT3 showed a Leu88Gln substitution in ParC. A variable decrease in the (fluoro)quinolone MICs was detected in the presence of the efflux pump inhibitor Phe-Arg-β-naphthylamide. PCR-based detection identified a qnrB gene in all three strains, which was chromosomally located (confirmed by plasmid transformation-conjugation experiments, Southern blot hybridization, S1 nuclease digestion, and sequence analysis). Analysis of a 3,629-bp segment (accession number AM774474) revealed the qnrB gene was located downstream of four open reading frames (pspF, pspA, pspB, pspC) encoding phage shock proteins, with similarity to Klebsiella pneumoniae plasmid pTN60013. The qnrB gene was a novel variant designated qnrB12, coding for a 215-amino acid protein with 98.9% nucleotide and 99.5% amino acid sequence identity to qnrB9 from Citrobacter freundii. Cloning qnrB12 into Escherichia coli TOP10 resulted in a 4-fold increase in nalidixic acid MIC and a 32-fold increase in ciprofloxacin MIC. Hybridization and sequence data suggest a qnrB12-carrying plasmid similar to pTN60013 was at least partially integrated into the chromosomal DNAs of these C. werkmanii strains.