Hybridoma technology was used to produce a set of monoclonal antibodies against a purified polypeptide fragment of type 24 streptococcal M protein to delineate the protective determinants of M protein exposed on the surface of the virulent streptococci. Several hybridoma antibodies were found to be opsonic against the homologous type streptococci. At least two of these antibodies (IIC3.7 and IIC4.6) protected mice against challenge infections with the homologous, but not a heterologous, serotype of bacteria. One of the hybridoma antibodies that reacted in high dilution (1:204,800) with the isolated M protein failed to react with the M protein on the surface of type 24 streptococci, and thus did not opsonize the homologous organisms or protect mice against challenge infections. Because hybridoma antibodies are directed against a single distinct immunodeterminant, these results indicate that protective immunity may be directed at any one of several distinct antigenic determinants of M protein exposed on the surface of virulent group A streptococci.