The aconitase (citB) gene of Bacillus subtilis is repressed during growth in a medium that contains a rapidly metabolizable carbon source and a source of 2-ketoglutarate. It is derepressed when either of these nutrient sources becomes limiting. Repression by rapidly metabolizable carbon sources was shown previously to depend at least in part on a DNA sequence located 67 to 84 base pairs upstream of the start point of citB transcription. In the present work, this region and surrounding DNA were mutagenized to identify more precisely the target for carbon catabolite repression. Mutations in a symmetric sequence located between positions -73 and -59 led to constitutive transcription from the citB promoter in media that normally provoke catabolite repression. By gel mobility shift assays, it was shown that at least one protein in extracts of B. subtilis binds to the symmetric sequence and that DNA of constitutive mutants binds to this protein much less effectively. A second sequence located near position -45 was also implicated in this regulation. A second form of regulation of citB was also investigated. This gene is known to be derepressed when cells are induced to sporulate by exhaustion of a nutrient broth medium or limitation of guanine nucleotide synthesis. The mutations that led to constitutivity with respect to the carbon source had no effect on citB expression in nutrient broth medium, indicating that control by catabolite repression and control by components of nutrient broth (presumably amino acids) act by different mechanisms.