We found that the N,6-O-diacetylmuramidase from Streptomyces globisporus (M-1) hydrolyzed the cell walls from Micrococcus lysodeikticus and Staphylococcus aureus. In contrast, hen egg white lysozyme (HEWL) was only able to hydrolyze the cell walls from M. lysodeikticus. 6-O-Acetylation of the muramoyl moieties, as found in the S. aureus cell walls, did not inhibit the activity of the M-1 enzyme whereas it was sufficient to inhibit HEWL. The disaccharide GlcNAc-MurNAc was not observed in the M. lysodeikticus cell wall hydrolyzate produced by the M-1, indicating that M-1 acts on the MurNAc moiety which are linked by peptides at the lactyl groups of the MurNAc moiety. M-1 displays both N-acetylmuramidase and N,6-O-diacetylmuramidase activity and has a different substrate specificity from HEWL.