A novel sporulation-control gene (spo0M) of Bacillus subtilis was cloned, sequenced and analyzed. The spo0M gene is located at the end of large tRNA gene clusters including rrnD and codes for a 257-amino-acid protein with a calculated size of 29.6kDa. The protein Spo0M has a strong negative charge (calculated pI=4.3) and shows no significant sequence homology to any known proteins. Gene disruption experiments revealed that spo0M is not essential for cell viability, but its disruption results in considerable impairments (decreasing by 20- to 100-fold) in sporulation. The morphological stage blocked in sporulation was stage 0 as observed by electron microscopy, and expression analysis using spo0Aps-bgaB fusion revealed an impaired gene expression of spo0A in the spo0M mutant. In contrast, spo0M disruption had no effect on antibiotic productivity. Propagation of the spo0M gene in wild-type cells using a high-copy-number plasmid also impaired sporulation, indicating that overproduction of Spo0M exerts certain negative effects on sporulation. spo0M gene expression is controlled by sigmaH, as demonstrated: (1) by monitoring expression of a bgaB transcriptional fusion integrated into the amyE locus on the chromosome of the wild-type or spo0H mutant cells, and (2) by in-vitro transcription of spo0M gene with EsigmaH.