The presence of interferon (IFN) has been demonstrated previously (i) in fluids obtained from the middle ears of children with Streptococcus pneumoniae infections, (ii) from the serum of mice injected intraperitoneally with either S. pneumoniae or Streptococcus pyogenes, and (iii) from human lymphoid cell cultures treated with a variety of bacteria. In this study, we showed that highly purified peptic extracts of three different serotypes of group A streptococcal M protein (pep M5, pep M6, and pep M24) stimulated human peripheral leukocytes to produce IFN. IFN production was apparent by 10 h and peaked 24 h after exposure. Dose-response experiments indicated that IFN could be detected in cultures treated with concentrations of M protein as low as 6 micrograms/ml, whereas maximum IFN production occurred at a concentration of 200 micrograms/ml. The IFN had antigenic and physicochemical characteristics of IFN-gamma. Preliminary leukocyte fractionation studies revealed that the IFN-producing cell was a nonadherent lymphocyte with receptors for sheep erythrocytes (T cell). Rabbit antisera specific for these structurally defined polypeptide fragments of streptococcal M protein (pep M5, pep M6, and pep M24) blocked IFN induction by each of the polypeptides. The data suggest that the different serotypes of streptococcal M protein may induce IFN by a common structural determinant shared by each of the polypeptide fragments tested.