Twenty-three multi-resistant strains of Escherichia coli were isolated at a single hospital in Beijing, China between January 1997 and May 1998. All isolates produced extended spectrum beta-lactamases (ESBLs) as detected by the double disk synergy test and the Etest ESBL strip (AB BIODISK, Solna Sweden). Additional antimicrobial susceptibility testing showed that most isolates were resistant to gentamicin, tobramycin, tetracycline, trimethoprim/sulfamethoxazole, ciprofloxacin, and cefepime. All isolates remained susceptible to imipenem with MICs of < or = 0.5 microgram/ml. The isolates each produced several beta-lactamases (range 1-4 enzymes/strain) with pI values ranging from 5.2-8.4. Molecular epidemiologic typing revealed four ribotypes and eight pulsed field gel electrophoresis (PFGE) patterns with subgroups among the 23 isolates. Clusters of isolates with the same DNA type were observed as follows (ribotype/PFGE): Wards A (242-5/2, and 242-5/3a), B (242-5/4), and C (880-1/1a). Moreover, similar molecular types were observed in patients from two or more different wards. Further use of isoelectric focusing results and co-resistance patterns produced evidence of potential nosocomial dissemination of strains in only two instances (two identical strains on one ward and two identical strains on different wards). There were also strong similarities in beta-lactamase pIs and co-resistances among many of the strains throughout this medical center. These data document the wide genetic diversity among E. coli producing ESBLs, and a potential for nosocomial spread of these highly resistant organisms requiring increasingly more sophisticated molecular-based techniques and local interventions.