Almost all clinical enterohaemorrhagic Escherichia coli (EHEC) O157:H7 isolates harbour a large virulence plasmid designated pO157. In this study, pO157 of EHEC O157:H7 reference strain EDL 933 was characterized at the molecular level. A restriction map was constructed by using seven restriction enzymes, with appropriate gel electrophoretic and hybridization methods. The molecular size of pO157 was determined to be 93.6 kb. By sequencing the DNA region extending in the 3'-direction of the previously described EHEC hlyC and hlyA genes, two further genes were discovered and analysed; these were termed EHEC hlyB and EHEC hlyD. The newly discovered genes together with the EHEC hlyC and hlyA genes constitute a typical RTX (Repeats in ToXin) determinant (EHEC hly operon) with the gene order CABD. The map position of the operon was determined by hybridization experiments. Analysis of a DNA fragment carrying the downstream flanking region of the EHEC hly operon revealed an open reading frame which was highly homologous to orf1 of RepFIB, a basic replicon of IncF plasmids. It was located close to the EHEC hly operon.