We developed a method that allows rapid isolation of mutant Bacillus subtilis 168 carrying an inversion of a specific DNA segment of the genome. Two incomplete neomycin resistance gene cassettes were integrated at both ends of the 1652-kb segment to be inverted. Reciprocal recombination within the 590-bp homologous region of these two cassettes created an intact neomycin resistance gene with concomitant inversion of the 1652-kb segment flanked by the two cassettes. Structure of the mutant genome was verified by analyzing the physical map for rare cutting endonucleases, SfiI, NotI, I-CeuI, and I-SceI. The inversion rate was estimated to be 6.9 +/- 1.4 x 10(-8)/cell/cell division at 37 degrees C. The method should be in principle applicable not only to other regions of the B, subtilis genome but also to other bacterial genomes.