The location of the stage 0 sporulation locus spo0F has been determined on a cloned fragment of Bacillus subtilis DNA. The spo0F gene and surrounding region was sequenced and was shown to code for a protein of Mr 14,286. The amino acid sequence of this deduced protein was 56% homologous to the amino-terminal domain of the spo0A gene product. The molecular weight of the Spo0F protein was approximately half that of the Spo0A protein, and its sequence was homologous to the amino-terminal half of the Spo0A protein. This same portion of the Spo0A protein showed ancestral relationship to the OmpR and SfrA regulatory proteins of Escherichia coli. Mutations in any of the genes encoding these proteins in either organism are highly pleiotropic and result in alterations in the regulation of membrane components, suggesting that they may have related roles in both organisms and that the stage 0 sporulation defect of spo0A and spo0F mutants is an indirect consequence of this regulatory system.