During bacteriophage studies of group A streptococci, some strains produced an inhibitory substance active against other group A strains. The inhibitory material was not found in broth cultures and could only be extracted from cultures grown in semi solid agar; its activity was assayed by the inhibition of growth and killing of a sensitive group A strain, 12203. Viable counts showed that 2 hr exposure to the factor at 37 °C produced greater than 90% inhibition and more than 60% killing of the treated culture. Turbidimetric measurements indicated no gross lysis of the sensitive culture and a dose response curve demonstrated that approximately two bactericidal factor molecules were sufficient to inactivate a colony forming unit of strain 12203. The inhibitory factor was of low molecular weight as judged by its diffusibility; it was concentrated by lyophilization and partially purified by gel filtration on G 10 Sephadex. The factor was resistant to proteolytic enzymes, nucleases and lipase as well as reducing and oxidizing conditions. Activity was lost at 60 °C, but the factor was stable at -70 °C. The bactericidal substance was stable within the pH range 2 to 12. These properties, as well as the wide range of activity of the bactericidal factor on several Gram positive and negative organisms, suggest that it is an inhibitory substance other than a bacteriocin.