The ribosomal RNA operons (rrn) of alkaliphilic Bacillus halodurans C-125 were characterized and compared with those of B. subtilis. We isolated clones containing rrn operons from a lambda phage library of the C-125 chromosome, and the complete nucleotide sequence of each was determined. Eight rrn operons were identified by PFGE analysis of the C-125 chromosome digested with I-CeuI. The transcriptional orientation of the rrn operons mapped on the chromosome by Southern hybridization analysis was the same as the direction of replication of the chromosome. These operons were designated as rrnA-H, starting from the oriC locus in clockwise rotation. Sequence and structural analyses of these operons suggested that six of the rrn operons in the C-125 chromosome, rrnA, rrnB, rrnC-rrnD, rrnE, and rrnH, correspond to rrnO, rrnA, rrnJ-rrnW, rrnI, and rrnD in B. subtilis, whereas the other rrn operons (rrnF and rrnG) were specifically observed in C-125. The rrn loci were positioned from 0 degrees to 90 degrees on the physical map, with the oriC locus assigned the position zero degrees. Two ORFs annotated as tnpA and ykfC, whose gene products are likely to act as transposases, were found downstream of these six operons. Comparative analysis of the 16S-23S and 23S-5S ITS (internally transcribed sequence) regions of B. halodurans C-125 and those of B. subtilis revealed that the ITS regions in C-125 were much longer than those in B. subtilis. There was no substantial difference in the length of potential promoter sequences in B. halodurans and B. subtilis.