Quinolone resistance is an emerging problem in China. To investigate the prevalence of the plasmid-mediated quinolone resistance genes qnr and aac(6')-Ib-cr, a total of 265 clinical isolates of Escherichia coli, Klebsiella pneumoniae, Citrobacter freundii, and Enterobacter cloacae with ciprofloxacin MICs of > or =0.25 microg/ml were screened at nine teaching hospitals in China. The qnrA, qnrB, qnrS, and aac(6')-Ib genes were detected by PCR. The aac(6')-Ib-cr gene was further identified by digestion with BtsCI and/or direct sequencing. The qnr gene was present in significantly smaller numbers of isolates with cefotaxime MICs of <2 microg/ml than isolates with higher MICs (> or =2.0 microg/ml) (20.6% and 42.1%, respectively; P < 0.05). aac(6')-Ib-cr was present in 17.0% of the isolates tested, and 7.9% of the isolates carried both the qnr and the aac(6')-Ib-cr genes. Among the isolates with cefotaxime MICs of > or =2.0 microg/ml, qnr and aac(6')-Ib-cr were present in 65.7% and 8.6% of E. cloacae isolates, respectively; 65.5% and 21.8% of K. pneumoniae isolates, respectively; 63.3% and 26.7% of C. freundii isolates, respectively; and 6.5% and 16.9% of E. coli isolates, respectively. The 20 transconjugants showed 16- to 128-fold increases in ciprofloxacin MICs, 14 showed 16- to 2,000-fold increases in cefotaxime MICs, and 5 showed 8- to 32-fold increases in cefoxitin MICs relative to those of the recipient due to the cotransmission of bla(CTX-M-14), bla(CTX-M-3), bla(DHA-1), bla(SHV-2), and bla(SHV-12) with the qnr and aac(6')-Ib-cr genes. Southern hybridization analysis showed that these genes were located on large plasmids of different sizes (53 to 193 kb). These findings indicate the high prevalence of qnr and aac(6')-Ib-cr in members of the family Enterobacteriaceae and the widespread dissemination of multidrug resistance in China.