The highly pleiotropic stage 0 sporulation locus of Bacillus subtilis, spo0A, has been cloned in bacteriophage lambda, subcloned in plasmids, and sequenced. The locus was found to code for a protein of 29,691 Da. Analysis of the in vivo transcripts from this region by nuclease S1 protection experiments located the start and stop of transcription of the locus. The transcription start site was preceded by a promoter resembling sigma 37-dependent promoters. Two mutations originally assigned to a second locus, spo0C, in this region because of their weakly pleiotropic phenotypes were cloned and sequenced. The mutations were found to be different missense alterations in the same base of the 10th codon preceding the carboxyl end of the Spo0A protein. These results, along with the finding that mutations in the spo0A gene product [Hoch, J. A., Trach, K., Kawamura, F. & Saito, H. (1985) J. Bacteriol. 161, 552-555] suppress the requirement for spo0B, spo0E, and spo0F gene products in transcription from sigma 28-dependent promoters, suggest that the Spo0A protein interacts directly with the transcription machinery to effect the initiation of sporulation. The deduced amino acid sequence of the Spo0A protein was highly related to that of the OmpR regulatory protein of Escherichia coli.