We reported previously the cloning and sequence of the Bacillus subtilis infB gene which encodes the essential IF2 factor required for initiation of translation (K. Shazand, J. Tucker, R. Chiang, K. Stansmore, H. U. Sperling-Petersen, M. Grunberg-Manago, J. C. Rabinowitz, and T. Leighton, J. Bacteriol. 172:2675-2687, 1990). The location of the 5' border of the infB operon was investigated by using integrative plasmids carrying various DNA fragments from the region upstream of the infB gene. The lethal effect of disruption of the infB transcriptional unit could be suppressed when the integrated plasmid introduced the spac promoter upstream of the infB operon and transformants were selected in conditions of induction of spac expression. Such an integrated plasmid was used as a starting point to clone the promoter of the infB operon. Primer extension mapping suggests that a single sigma A-type promoter controls transcription of the infB operon. The sequence of a 5,760-bp region encompassing the infB gene was determined. The infB operon is located immediately downstream of the polC gene and comprises seven open reading frames, four of which appear to be the homologs of genes present in the same order in the Escherichia coli infB operon, including nusA. The striking similarity between the E. coli and B. subtilis infB operons suggests that the function of each gene pair is conserved and that the B. subtilis NusA homolog, which is 124 residues shorter than its E. coli counterpart, could play a role similar to its role in E. coli.