The complete nucleotide sequence and organization of the enteropathogenic Escherichia coli (EPEC) adherence factor (EAF) plasmid of EPEC strain B171 (O111:NM) were determined. The EAF plasmid encodes two known virulence-related operons, the bfp operon, which is composed of genes necessary for biosynthesis of bundle-forming pili, and the bfpTVW (perABC) operon, composed of regulatory genes required for bfp transcription and also for transcriptional activation of the eae gene in the LEE pathogenicity island on the EPEC chromosome. The 69-kb EAF plasmid, henceforth designated pB171, contains, besides the bfp and bfpTVW (perABC) operons, potential virulence-associated genes, plasmid replication and maintenance genes, and many insertion sequence elements. Of the newly identified open reading frames (ORFs), two which comprise a single operon had the potential to encode proteins with high similarity to a C-terminal region of ToxB whose coding sequence is located on pO157, a large plasmid harbored by enterohemorrhagic E. coli. Another ORF, located between the bfp and bfpTVW operons, showed high similarity with trcA, a bfpT-regulated chaperone-like protein gene of EPEC. Two sites were found to be putative replication regions: one similar to RepFIIA of p307 or F, and the other similar to RepFIB of R100 (NR1). In addition, we identified a third region that contains plasmid maintenance genes. Insertion elements were scattered throughout the plasmid, indicating the mosaic nature of the EAF plasmid and suggesting evolutionary events by which virulence genes may have been obtained.