A cell wall hydrolase homologue, Bacillus subtilis YddH (renamed CwlT), was determined to be a novel cell wall lytic enzyme. The cwlT gene is located in the region of an integrative and conjugative element (ICEBs1) and a cwlT-lacZ fusion experiment revealed the significant expression when mitomycin C is added to the culture. Judging from the Pfam database, CwlT (cell wall lytic enzyme T [Two-catalytic domains]) has two hydrolase domains which exhibit high amino acid sequence similarity to D,L-endopeptidases and relatively low similarity to lytic transglycosylases at the C- and N-terminals, respectively. The purified C-terminal domain of CwlT (CwlT-C-His) could hydrolyze the linkage of D--glutamyl-meso-diaminopimelic acid in B. subtilis peptidoglycan, suggesting that the C-terminal domain acts as a D,L-endopeptidase. On the other hand, the purified N-terminal domain (CwlT-N-His) could also hydrolyze the peptidoglycan of B. subtilis. However, on reverse-phase HPLC and mass spectrometry (MS) and MS-MS analyses of the reaction products by CwlT-N-His, this domain was interestingly determined to act as an N-acetylmuramidase not a lytic transglycosylase. Moreover, the site-directed mutagenesis analysis revealed that Glu-87 and Asp-94 are sites related with the cell wall lytic activity. Since the amino acid sequence of the N-terminal domain of CwlT exhibits low similarity to those of the soluble lytic transglycosylase (SLT) and muramidase (goose lysozyme), this domain represents “a new category of cell wall hydrolases”.