A restriction enzyme map was constructed of a 3.3-kb plasmid derived from enterohemorrhagic Escherichia coli O157:H7 strain 4821 using the enzymes SmaI, HpaI, FokI, HaeIII, StyI, RsaI, Bg/II, ClaI, and EcoRV. The molecular size of p4821 was 3307 bp, determined by nucleotide sequencing. Homology searches in the EMBL database library revealed that the nucleotide sequence of P4821 is similar (> 98%) to the core region of the antibiotic resistance plasmid NTP16 of Salmonella typhimurium strains. Nucleotide sequence analysis showed that p4821 contains all the information necessary for its replication, stability, and mobilization. However, the lack of tra genes indicates that the plasmid is nonconjugative. A possible transposon insertion site was found in p4821 but two such sites were found at the boundaries between the core region and the antibiotic resistance encoding transposons in plasmid NTP16. Using a hybridization assay, we determined that of 50 E. coli O157:H7 strains isolated in 1996 in Würzburg, Germany from patients with diarrhea and hemolytic-uremic syndrome, 4 strains (8%) contained plasmid p4821-specific sequences.