The sequence of a 4.4 kbp region of DNA from Bacillus subtilis 168, lying between sporulation genes spoVD and spoVE, has been determined as part of the B. subtilis genomic sequencing programme. The region contains three genes with high sequence similarity to the murE, mraY and murD genes of Escherichia coli. The products of these genes are likely to catalyse various steps in the formation of the precursors for peptidoglycan synthesis in B. subtilis. The regions at 133 degrees on the standard genetic map of the B. subtilis chromosome, and in the 2 min region of the E. coli genetic map, are now shown to contain a large cluster of functionally related genes. Although the linear order of the genes in the cluster is conserved, three genes that are present in the E. coli chromosome, and which are likely to be essential for peptidoglycan synthesis in both organisms, are absent from this region of the B. subtilis chromosome. In general, the B. subtilis cluster differs from that of E. coli in having more extensive intergenic regions, with less potential for translational coupling.