A new peptidoglycan hydrolase, Bacillus subtilis YojL (cell wall-lytic enzyme associated with cell separation, renamed CwlS), exhibits high amino acid sequence similarity to LytE (CwlF) and LytF (CwlE), which are associated with cell separation. The N-terminal region of CwlS has four tandem repeat regions (LysM repeats) predicted to be a peptidoglycan-binding module. The C-terminal region exhibits high similarity to the cell wall hydrolase domains of LytE and LytF at their C-terminal ends. The C-terminal region of CwlS produced in Escherichia coli could hydrolyze the linkage of d-gamma-glutamyl-meso-diaminopimelic acid in the cell wall of B. subtilis, suggesting that CwlS is a d,l-endopeptidase. beta-Galactosidase fusion experiments and Northern hybridization analysis suggested that the cwlS gene is transcribed during the late vegetative and early stationary phases. A cwlS mutant exhibited a cell shape similar to that of the wild type; however, a lytE lytF cwlS triple mutant exhibited aggregated microfiber formation. Moreover, immunofluorescence microscopy showed that FLAG-tagged CwlS was localized at cell separation sites and cell poles during the late vegetative phase. The localization sites are similar to those of LytF and LytE, indicating that CwlS is involved in cell separation with LytF and LytE. These specific localizations may be dependent on the LysM repeats in their N-terminal domains. The roles of CwlS, LytF, and LytE in cell separation are discussed.