Staphylococcus aureus FDA 209P produces two extracellular bacteriolytic enzymes, 51-kDa endo-beta-N-acetylglucosaminidase (GL) and 62-kDa N-acetylmuramyl-L-alanine amidase (AM), both of which can disperse cell clusters. To characterize the physiological roles of these enzymes in vivo, mutants with altered autolysin activity were isolated, and their degree of cluster formation in broth culture was assessed. Bacteriolytic activities of GL and AM, produced and secreted from these mutants into the culture fluid and detected with activity gels, coincided well with the degree of cluster formation of the mutants. The mutants with little or no enzyme activity grew in clusters, whereas those with high activity grew as well-separated cocci, suggesting that these enzymes are involved in cell separation of S. aureus in vivo.