We have identified the transcription start site and regulatory region governing the expression of a sporulation gene in the spoIVC locus of Bacillus subtilis. Efficient expression and developmental regulation of this gene was controlled from a promoter region that extended no more than 110 base pairs upstream and no more than 4 base pairs downstream from the start site of transcription, on which basis we infer that spoIVC is regulated at the level of transcription initiation. Using a transcriptional fusion of the spoIVC gene to the lacZ gene of Escherichia coli, we found that spoIVC expression was turned on at the third to fourth hour of sporulation (at about the developmental stage [IV] that its products are required in spore formation) and that this transcription was largely restricted to the mother cell chamber of the sporangium. Mutations in many different spo genes (causing blocks at stages 0 to V) were found to influence (negatively and positively) the level of spoIVC expression. Our results distinguish the mode of spoIVC regulation from that of previously studied sporulation genes and indicate that it is representative of a new regulon of mother cell-specific gene expression.