Biochemical and physical properties of the pure bacteriolytic enzymes excreted by three different Staphylococcus species (S. aureus, S. simulans, and S. saprophyticus) were investigated. Although the three enzymes have previously been shown to share the same specificity of action (endo-beta-N-acetylglucosaminidase activity), their biochemical features clearly indicated that they were three different enzymes, confirming what had previously been suggested by the different lytic-activity patterns displayed by each species and the different procedures needed to achieve purification of each enzyme. Very different values resulted from molecular weight determination: 80,000 for the S. aureus enzyme, 45,000 for the S. simulans enzyme and 31,000 for the S. saprophyticus enzyme. Other important differences were observed in their kinetics of activity on Micrococcus luteus purified cell walls; their stability; their bacteriolytic spectrum against heat-killed cells of various microorganisms; and their response to physical and chemical factors, such as temperature, pH, ionic strength, divalent cations, chelating agents, thiol compounds, and glucose derivatives.