An I-CeuI-NotI-SfiI endonuclease map of the Bacillus subtilis 166 genome was constructed. It was almost identical to that of B. subtilis 168 except for the inversion of an approximately 1900 kb DNA segment, the translocation of an approximately 100 kb segment and the duplication of a 5 kb segment. Continuity of the inverted segment was investigated by direct measurement of the distances between the two genomic loci where I-SceI recognition sites were created in the 168 and the 166 genomes. Size difference of the I-SceI fragments between the two strains fully demonstrated the inversion of an approximately 1900 kb long 'continuous' DNA segment and the 'location' of the two inversion junctions in the genome. The 100 kb DNA segment including the lysogenic SP beta prophage was translocated close to one of the inversion junctions and was probably associated with the duplication of a 5 kb segment. These rearrangements are consistent with those indicated by genetic analyses.