Bacillus subtilis yvcE (named cwlO) encodes a polypeptide consisting of 473 amino acid residues, and the N-terminal region contains a putative signal sequence and the C-terminal region exhibits high similarity to those of the NLPC/P60 superfamily (DL-endopeptidase family II). Northern blotting and cwlO-lacZ fusion analyses indicated that the cwlO gene is expressed as a monocistronic mRNA during the vegetative growth phase. The C-terminal region of CwlO was cloned into an Eseheriehia coli histidine-tagged vector and the protein, C-CwlO-6His, was produced in E. coli. The purified C-CwlO-6His protein exhibited cell wall hydrolase activity and the substrate bond specificity indicated that it is a DL-endopeptidase. The cell wall hydrolytic activity which seems to be derived from partially degraded CwlO (YvcE) was found in the culture supernatant of B. subtilis, but not in the cell wall binding fraction. The disruption of cwlO did not result in any difference in cell growth, morphology, or motility.