The cytotoxic moiety(ies) in highly purified streptococcal M protein has been shown to be distinct from the type-specific M determinant. This nontype-specific M-associated determinant(s) (NTSM) causes humoral and cellular immunotoxic responses in man. NTSM is common to M protein prepared from all streptococcal serotypes studied so far. In this study, immunoabsorbents prepared by entrapping purified M proteins in polyacrylamide gel were employed to identify, separate, and purify antibodies directed against NTSM as well as against the type-specific M (TSM) determinants. We found that anti-NTSM present in human blood mediated cytotoxic platelet and leukocyte reactions in the presence of "M proteins" prepared from groups A, C, and G streptococci. Human sera that produced cytotoxic reactions and fixed complement in the presence of highly purified M protein but that contained no antibody to the homologous TSM determinant were used as a source of anti-NTSM. Anti-NTSM was absorbed with and eluted from M protein-polyacrylamide particles and identified as IgG. Antibodies to NTSM were present in most normal human sera and some primate sera (rhesus monkey and baboons) but not in the sera of other common laboratory animals. Further absorption studies showed NTSM to be a component not only of extractable M protein but also of protoplast membranes and of cell walls of avirulent streptococci that lacked extractable TSM antigen. Preparation of antisera that can distinguish between the type-specific protective moiety and the closely associated immunotoxic components in purified M protein vaccines may help answer whether or not M-associated moieties play a role in pathogenesis of poststreptococcal diseases.