The peptidoglycan hydrolyzing endo-beta-N-acetylglucosaminidase of Staphylococcus simulans 22 is not able to attack intact cell walls of S. simulans 22, but hydrolyzes cell walls of Micrococcus luteus and soluble peptidoglycan chains of S. simulans 22. Hydrolysis of cell walls of M. luteus is activated in presence of organic cations such as poly-L-lysine (n = 17) and the peptide antibiotics Pep 5 and nisin, whereas hydrolysis of soluble peptidoglycan chains is not influenced. High concentrations of inorganic cations inhibit enzyme activity. These effects are discussed with respect to the cationic nature of the enzyme (pI greater than 9.5) and the regulation of the concerted action of the N-acetylmuramoyl-L-alanine amidase and the glucosaminidase during S. simulans 22 autolysis in vivo.