The promoter (amyP) of the Bacillus subtilis alpha-amylase gene, which is recognized by E sigma A, has a three out of six match to the consensus promoter in both the -35 and -10 hexamers. Oligonucleotide-directed mutagenesis was used to identify important bases for promoter utilization in the spacer sequence between the hexamers. Mutations in the sequence TGTG extending from positions -18 to -15 (the -16 region) caused a 5-94-fold decrease in alpha-amylase production. A G-C transversion at position -15 was the most detrimental mutation: it essentially eliminated amyP utilization in B. subtilis and in Escherichia coli. Mutating the -35 and -10 hexamers to the E sigma A consensus promoter increased alpha-amylase production 56-fold in B. subtilis and fivefold in E. coli. Introducing the -15 G to C transversion into the consensus promoter reduced alpha-amylase production threefold, in contrast to the 94-fold reduction for the wild-type promoter in B. subtilis. The -15 G to C transversion did not reduce alpha-amylase synthesis directed by the consensus promoter in E. coli. The alpha-amylase gene is subject to two forms of transcriptional regulation: catabolite repression and temporal regulation. None of the mutants constructed in this study had any effect on either type of regulation. The -16 region, especially the G at position -15, appears to be moderately conserved in B. subtilis and in other Gram-positive organisms and weakly conserved in E. coli. The evidence suggests that the -16 region is an additional region of E sigma A promoters in B. subtilis and E sigma 70 promoters in E. coli, essential in some weak promoters such as the alpha-amylase promoter but, of little benefit to very strong promoters.