The phytopathogenic bacterium Agrobacterium tumefaciens is unique in that it possesses both linear and circular DNA chromosomes in addition to a plant-tumor-inducing (Ti) plasmid. We analyzed the two chromosomal DNA molecules in strain MAFF301001, whose Ti plasmid has already been sequenced completely. Physical maps of the chromosomal DNAs were constructed by Southern hybridization experiments using Pme I and Swa I fragments and short fragments bridging the Swa I fragments with special care to avoid any missing fragment. Hybridization with 16S rDNA probe showed one rDNA locus on the linear chromosome and two loci on the circular chromosome. For this bacterium to be pathogenic, not only Ti plasmid but also chromosomal genes are required. The chromosomal virulence (chv) genes (chvA, chvB, chvD, chvE, chvG, chvH, and chvI) and the chromosomal genes affecting the virulence [acvB, pgm(exoC), glgP, miaA, and ros] were successfully mapped onto 5 different regions in the chromosomal physical maps. These chv genes and the chromosomal genes affecting the virulence other than pgm and glgP were found on the circular chromosome, whereas the pgm and glgP genes were located on the linear chromosome. In contrast to the large terminal inverted repeats of Streptomyces linear chromosomal DNA, no hybridization signal was detected between left and right terminal fragments of the linear A. tumefaciens chromosome. Quantitative analysis of DNA fragments indicated that the copy numbers of the two chromosomal DNAs and the Ti plasmid are identical.