The ability of macromolecules to cross the capsular layer of encapsulated microorganisms and interact with their cell walls is important in considerations of the mechanisms of resistance to phagocytosis and of antigen masking in such strains. Lysostaphin was employed as a probe of the penetrability of the Staphylococcus aureus capsule. The rates of lysostaphin-induced lysis of encapsulated and unencapsulated S. aureus strains were compared. Encapsulated S. aureus strains M and Smith diffuse were lysed by lysostaphin at the same rate as their respective unencapsulated counterpart strains M variant and Smith compact. Growth of the M strain in a medium designed to enhance capsule production did not delay the onset or decrease the rate of lysis of the strain compared with organisms grown in normal medium. Cations did not selectively decrease the rate of lysis of the encapsulated strain, but inhibited the lysis of both the M and M variant strains. Peptidoglycan, the presumed lysostaphin target, isolated from both M and M variant strains was digested by lysostaphin at very similar rates. In contrast to whole cells, cations stimulated the rate of lysostaphin digestion of peptidoglycan. It is concluded that the fraction of lysostaphin active in cell lysis, believed to be a glycylglycine endopeptidase with a molecular weight of about 25,000, passes freely through the capsular layer to its target in the staphylococcal cell wall.