All the SfiI sites and most of the NotI sites were located precisely on the chromosome of Bacillus subtilis 168 by a novel method, termed gene-directed mutagenesis. The stepwise elimination of these restriction sites by this method allowed not only the physical connection of the restriction fragments but also the accurate determination of the position of the restriction sites themselves. The resulting physical map of the 4165 x 10(3) base-pair B. subtilis chromosome has been correlated with the genetic map by determination of the exact location of known genes. The complete physical map provides a rapid and accurate way for mapping of new genes as well as analysis of large DNA rearrangements on the chromosome. The novel strategy is, in principle, applicable to the analysis of the genome of other organisms.