Carbapenems such as imipenem and meropenem are first-line agents for the treatment of serious nosocomial infections caused by multidrug-resistant clinical isolates of bacteria belonging to the family Enterobacteriaceae. However, resistance to carbapenems has increased dramatically among members of the family Enterobacteriaceae isolated from a teaching hospital in Shanghai, China. In the present study, we investigated the prevalence and molecular characteristics of carbapenem-resistant clinical isolates of Enterobacteriaceae. None of the 77 clinical isolates collected from 2002 to 2009 were susceptible to ertapenem and only 6.5 % and 1.3 % of isolates were susceptible to imipenem and meropenem, respectively. Colistin and tigecycline were found to be the most active agents against carbapenem-resistant Enterobacteriaceae isolates, inhibiting 90 % of isolates at a concentration of 1 µg ml(-1) and 4 µg ml(-1), respectively. The results of PFGE analysis suggested that many of the KPC-2-producing isolates of Citrobacter freundii and Klebsiella pneumoniae were clonally related. Most of these isolates were isolated from the same ward, namely the neurosurgical ward, suggesting horizontal transfer of the KPC-2-encoding gene in these isolates. Of the 77 isolates, 84.4 % were found, by PCR, to be capable of carbapenemase production. SDS-PAGE analysis revealed that 75.3 % (58/77) of the isolates had lost at least one porin protein. Our results suggested that the prompt detection of carbapenemase-producing strains is critical for the containment of nosocomial transmission. As no novel antimicrobials have been identified for use in the treatment of these pan-drug-resistant isolates, further studies should focus on the rational use of available antibiotics, the implementation of active antibiotic resistance surveillance and the strict implementation of infection control measures to avoid the rapid spread or outbreak of carbapenemase-producing Enterobacteriaceae in health-care facilities.