Southern hybridization analysis of Bacillus subtilis 168S chromosomal DNA with a Bacillus licheniformis cell wall hydrolase gene, cwlM, as a probe indicated the presence of a cwlM homolog in B. subtilis. DNA sequencing of the cwlM homologous region showed that a gene encoding a polypeptide of 255 amino acids with a molecular mass of 27,146 Da is located 625 bp upstream and in the opposite direction of spoVJ. The deduced amino acid sequence of this gene (tentatively designated as cwlC) showed an overall identity of 73% with that of cwlM and of 40% with the C-terminal half of the B. subtilis vegetative autolysin, CwlB. The construction of an in-frame cwlC-lacZ fusion gene in the B. subtilis chromosome indicated that cwlC is induced at 6 to 7 h after sporulation (t6 to t7). The spoIIIC (sigma K) mutation and earlier sporulation mutations greatly reduced the expression of the cwlC-lacZ fusion gene. Northern hybridization analysis using oligonucleotide probes of the cwlC region indicated that a unique cwlC transcript appeared at t7.5 and t9. Transcriptional start points determined by primer extension analysis suggested that the -10 region is very similar to the consensus sequence for the sigma K-dependent promoter. Insertional inactivation of the cwlC gene in the B. subtilis chromosome caused the disappearance of a 31-kDa protein lytic for Micrococcus cell walls, which is mainly located within the cytoplasmic and membrane fractions of cells at t9. The CwlC protein hydrolyzed both B. subtilis vegetative cell walls and spore peptidoglycan.