We present definitive evidence that at least one protective antigenic determinant on type 5 M protein of group A streptococci evokes antibody that is cross-reactive with human heart tissue. One of nine rabbits immunized with a peptide fragment of type 5 M protein (pep M5) produced antibody that cross-reacted by immunofluorescence with sarcolemmal membranes of human heart. The cross-reactive antibody could be removed by absorbing the antiserum with sarcolemmal membranes, types 5 and 19 streptococci, or their pepsin-extracted M proteins, but with no other serotypes tested. Although each of the pep M5 immune sera was opsonic for type 5 streptococci, only the heart-reactive antiserum opsonized type 19 streptococci. The opsonization of type 19 streptococci was abolished by absorbing the antiserum with sarcolemmal membranes isolated from human heart tissue. Purified heart-reactive antibodies eluted from sarcolemmal membranes opsonized both types 5 and 19 streptococci, indicating that the heart cross-reactive determinant of type 5 M protein is cross-protective. The cross-reactive antigen was purified by affinity chromatography from detergent extracts of sarcolemmal membranes and determined to be a complex protein composed of four subunits apparently linked by disulfide bonds.