The 30-kDa sporulation-specific peptidoglycan hydrolase CwlC of Bacillus subtilis 168 was purified and characterized. It is an N-acetylmuramoyl-L-alanine amidase (amidase) that is associated with the mother cell wall of sporulating cells, and although it is secreted, it undergoes no N-terminal processing except removal of the initial methionine. It was found that mother cells of a strain insertionally inactivated in cwlC and lytC (the major vegetative amidase gene) did not lyse at the end of sporulation. Mutants with single mutations in cwlC or lytC lysed, and so the two autolysins must have mutually compensatory roles in mother cell lysis. Active CwlC and LytC are present at the time of mother cell lysis; however, reporter gene analysis revealed that lytC transcription ceases early in sporulation, and therefore the function that LytC has in mother cell lysis is performed by material remaining from presporulation expression. Autolytic enzymes similar in molecular mass to CwlC were detected in two other Bacillus species by their cross-reactivity with anti-CwlC antiserum.