A number of epitope specificities associated with the cell wall polysaccharide antigen of group A streptococci were identified in a polyclonal rabbit antiserum induced in rabbits by whole group A streptococci and in polyclonal convalescent human antisera from children that had recovered from streptococcal A infections. The identification was achieved by using a series of synthetic oligosaccharides, glycoconjugates, and bacterial polysaccharide inhibitors to inhibit the binding of the group A helical polysaccharide to the polyclonal antisera. The exclusively dominant epitope expressed in the convalescent human antisera was the doubly branched extended helical hexasaccharide with the structure alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap. The hexasaccharide epitope also bound with the highest immunoreactivity to the rabbit antiserum. In contrast, the human antisera did not show significant binding to the singly branched pentasaccharide with the structure alpha-L-Rhap(1-->2)alpha-L-Rhap(1-->3)alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-L-Rhap or the branched trisaccharide alpha-L-Rhap(1-->2)[beta-D-GlcpNAc(1-->3)]alpha-l-Rhap, although both these haptens bound significantly to the same rabbit antiserum, albeit with less immunoreactivity than the hexasaccharide. Inhibition studies using streptococcal group A and B rabbit antisera and the inhibitors indicated above also suggested that the group A carbohydrate, unlike the group B streptococcal polysaccharide, does not contain the disaccharide alpha-L-Rhap(1-->2)alpha-L-Rhap motif at its nonreducing chain terminus, stressing the importance of mapping the determinant specificities of these two important streptococcal subcapsular group polysaccharides to fully understand the serological relationships between group A and group B streptococci.