The program of gene expression that underlies endospore formation by Bacillus subtilis may be controlled in part by a sporulation-induced form of RNA polymerase, E sigma 29. The nucleotide sequences of four promoters, which are known to be recognized by E sigma 29, are highly conserved at two regions, 10 bp and 35 bp upstream from the start point of transcription. We have used oligonucleotide-directed mutagenesis to construct several base substitutions in the ctc promoter from B. subtilis to test the role of the highly conserved sequences in utilization of the promoter by E sigma 29. In vitro transcription analysis demonstrated that the conserved nucleotides at positions -15, -14 and -12 affect the utilization of the promoter by E sigma 29. These and previous results support a model in which E sigma 29 recognizes its cognate promoters by interacting with nucleotides near the -10 and -35 regions. We also examined the effects of these base substitutions on utilization of the promoter by two other forms of RNA polymerase from B. subtilis, E sigma 37 and E sigma 32.