Previous studies have shown that the amino-terminal regions of group A streptococcal M proteins contain primarily protective (opsonic) epitopes and not tissue-cross-reactive epitopes. Limited primary structures from multiple serotypes of M protein containing only protective epitopes could potentially be linked together to form a broadly protective vaccine. The present studies were undertaken to determine the protective immunogenicity of a recombinant, multivalent hybrid molecule containing amino-terminal subunits of types 24, 5, 6, and 19 M proteins. Polymerase chain reaction primers were designed to amplify emm gene fragments ranging from 35 to 113 codons. The PCR products were ligated in tandem and inserted into pKK223-3. The tetravalent M protein that was purified from extracts of Escherichia coli migrated as a single band on SDS-PAGE with an apparent m.w. of 31 kDa. In immunoblot analyses, the hybrid protein reacted with serotype-specific antisera indicating that it contained all four M protein subunits. Rabbits immunized with the purified tetravalent M protein developed significant antibody levels against all four serotypes of native M proteins represented in the hybrid protein. None of the antisera cross-reacted with human tissues. The immune sera also opsonized all four serotypes of group A streptococci. Our data show that a hybrid protein containing subunits from multiple M proteins can evoke broadly protective immune responses without tissue-cross-reactive antibodies.