Transcription of the Bacillus subtilis spo0E gene is controlled by the AbrB transition state regulator. In AbrB+ strains, a single transcript, P1, was observed for the spo0E gene. In an abrB4 mutant strain, a second transcription start site 3 bases upstream from P1 was found to be used for the predominant transcript. P1 transcription was insensitive to the state of the abrB gene. Mutants carrying deletion or antibiotic cassette insertion mutations in the spo0E gene were Spo+. Multiple copies of the spo0E gene, not just the promoter region, were found to render strains incapable of sporulation. Spo+ strains that arose spontaneously from such Spo- strains were found to have deletions in the spo0E coding sequence on the plasmid. Strains carrying a deletion of the spo0E gene segregated Spo- colonies. These colonies were found to have secondary mutations in or near the spo0A, spo0B, or spo0F gene, suggesting that deletion of the spo0E gene results in increased pressure to sporulate that is compensated for by inactivation of one or more of the components of the signal transduction system leading to the initiation of sporulation. spo0E deletions were suppressors of the spo0F221 missense mutation but had no effect on the regulation of the spo0F, kinA, spo0A, or spo0B genes. The results suggest that the spo0E gene product is a negative regulator of the signal transduction pathway leading to sporulation.