A family outbreak of enterohaemorrhagic Escherichia coli (EHEC) O157:H7 infection occurred in October 2003 in the Hiroshima prefecture, Japan. Four isolates of EHEC O157:H7, 03064, 03065, 03066 and 03067, were recovered from a 1-year-old daughter, mother, father and 3-year-old daughter, respectively. All EHEC O157:H7 isolates were positive for Stx1 and Stx2 Shiga toxins. Surprisingly, DNA fingerprinting profiles obtained by PFGE showed that the first isolate, 03064, had unique XbaI and BlnI profiles that differed from the other three isolates. Also, plasmid analysis results revealed that isolate 03064 contained an extra plasmid larger than the classic large plasmid of EHEC O157, pO157 (93.6 kb). This new plasmid was named pMDR157. Furthermore, isolate 03064 showed a multidrug-resistance (MDR) phenotype against streptomycin, spectinomycin, co-trimoxazole (trimethoprim/sulfamethoxazole), ampicillin and tetracycline; the other isolates were completely sensitive to these antibiotics. Molecular analysis of the MDR phenotype in this unique strain revealed the presence of a class 1 integron containing two gene cassettes: a dihydrofolate reductase type 1 gene (dfrI), which confers resistance to trimethoprim, and an aminoglycoside adenyltransferase gene (aadA1), which confers resistance to streptomycin and spectinomycin. Southern blot hybridization showed that the class 1 integron was located in the extra plasmid, pMDR157. The ampicillin resistance was found to be due to the presence of the TEM-1-type beta-lactamase gene. The MDR phenotype was transferred successfully to E. coli HB101 by conjugation, indicating that both the class 1 integron and the TEM-1 beta-lactamase were located on the conjugative transferable plasmid, pMDR157. To the authors' knowledge, this is the first report of the identification of a beta-lactamase gene in EHEC O157.