We have determined a 180 kb contiguous sequence in the replication origin region of the Bacillus subtilis chromosome. Open reading frames (ORF) in this region were unambiguously identified from the determined sequence, using criteria characteristic for the B. subtilis gene structure, i.e., starting with an ATG, GTG or TTG codon preceded by sequences complementary to the 3' end of the 16S rRNA. Four rRNA gene sets, 7 individual tRNA genes and 1 scRNA gene were identified, occupying 20 kb in total. In the remaining 160 kb region, 158 ORFs were identified, suggesting that 1 ORF is coded on average by 1 kb of DNA of the B. subtilis genome. Among the 158 ORFs, the functions of 48 ORFs were assigned and those of 11 ORFs are suggested through significant similarities to known proteins present in data banks. However, the functions of more than half of the ORFs (63%) remain to be determined.