The spo0F gene of Bacillus subtilis encodes a protein that functions as a secondary messenger in a phosphorelay system controlling the initiation of sporulation. Transcription of the spo0F gene was known to be dependent on an intact gene for the transcription regulator Spo0A. In vitro footprint analysis revealed that Spo0A protein bound to two locations in the spo0F promoter region. Deletion of a 40 bp region upstream of one of the promoters (P2) abolished the activation of spo0F expression that occurs at the onset of stationary phase and sporulation. This 40 bp region contains a Spo0A-binding site. These observations are consistent with a hypothesis that Spo0A binding to this region is responsible for activating spo0F transcription. Additionally, Spo0A binding at a downstream site could modulate the level of this activation. Since Spo0F protein is required for the formation of Spo0A-P (the form needed for transcriptional activation) a positive feedback loop controls transcription of spo0F.