A physical map of the chromosome of Dichelobacter nodosus strain A198 was constructed using the restriction endonucleases EagI and StuI. Mapping data indicated the presence of a single, circular chromosome of 1.54 Mb. The three rRNA operons and the virulence related locus (vrl) were precisely positioned at the junctions of EagI and StuI fragments, and their transcriptional orientations were also determined. Other D. nodosus genes were assigned to specific EagI and StuI fragments. Analysis of the resultant map revealed that the putative virulence genes were not clustered on the chromosome which suggests that the D. nodosus virulence determinants have been acquired gradually and that virulence in D. nodosus is an evolving trait.