Xanthomonas campestris is an important plant pathogenic bacterium which causes severe diseases in a wide variety of plant species. We have generated a macrorestriction map of the X. campestris ( axonopodis) pv. glycines chromosome employing pulsed-field gel electrophoresis (PFGE). Restriction endonucleases PacI (5′-TTAATTAA), PmeI (5′-GTTTAAAC) and SwaI (5′-ATTTAAAT) digested the chromosomal DNA into three, five, and five fragments, respectively. In addition, intron-encoded restriction endonuclease I- CeuI was employed to locate the position of the 23S rRNA genes ( rrlA and rrlB). All of the generated restriction fragments were aligned along the chromosome using multiple restriction enzyme digestion and two-dimensional PFGE (2-D PFGE) in conjunction with Southern hybridization analysis. This physical map construction has revealed a single circular chromosome with a size of approximately 5 Mb. Two rRNA genes were localized on the chromosome map. Several genes involved in pathogenesis ( xpsD, opsX, and pat) as well as genes involved in the biosynthesis of xanthan gum ( xanAB, rfbCDAB) were also localized.