A new peptidase which splits substrates related to the peptidic chains of peptidoglycans was found in the cell cytoplasm of sporulating Bacillus sphaericus. This is a gamma-D-glutamyl-L-diaminoacid endopeptidase (endopeptidase II). It was shown to have substrate requirements different from those of the previously described gamma-D-glutamyl-(L)meso-diaminopimelate endopeptidase (endopeptidase I). The substrates for endopeptidase II are peptides of the general type: formula: (see text). Unsubstituted N-terminal L-alanine was a strict requirement for endopeptidase II activity. Specific activities were variable with the nature and the substitution of the diaminoacid C-terminal groups. The role of endopeptidase II in the biosynthesis of the spore cortex is discussed.