In previous studies, encapsulated Staphylococcus aureus strains have been shown to resist phagocytosis. In this investigation, the nature of the interference with phagocytosis by human polymorphonuclear leukocytes was examined by studying the opsonization of two pairs of unencapsulated (Smith compact and M variant) and encapsulated (Smith diffuse and M) S. aureus strains. The uptake of [3H]glycine-labeled bacteria by normal leukocytes was quantitatively measured after incubation of bacteria in pooled serum, C2-deficient serum, immunoglobulin-deficient serum, and serum from a rabbit immunized with S. aureus M. The presence of a capsule was found to interfere with opsonization by both the classical and alternative pathways of complement as well as by heat-stable opsonic factors in nonimmune human serum. This interference was significantly greater in the case of the S. aureus M strain than in the case of the Smith diffuse strain. The only effective opsonic source for S. aureus M was immune rabbit serum. It is proposed that encapsulation of S. aureus strains interferes with phagocytosis by preventing effective bacterial opsonization.