The pleiotropic stage 0 sporulation locus spo0E was isolated and sequenced. The spo0E gene was found to code for a protein of 9791 molecular weight. Two spo0E mutations were identified by sequence analysis and were found to give rise to nonsense codons within the gene. The results indicated that it is the lack of the spo0E gene product that is responsible for the sporulation-defective phenotype. The DNA fragment containing the spo0E locus was inhibitory to sporulation when present on a multicopy plasmid. Since DNA fragments containing only the upstream region of the gene were also inhibitory, this effect was not due to over-production of the spo0E gene product. Coupling the transcription of the spo0E gene to beta-galactosidase in an integrative plasmid vector revealed that active transcription of this gene begins at the end of exponential growth and continues through the early part of sporulation. Studies of the regulation of this gene have allowed the generation of a hypothesis to explain the interactions of those five stage 0 genes involved in the activation of sporulation-specific transcription.