The R antigen, a trypsin-resistant protein observed in group A, C, F, G, and L streptococci, has also been found in group B streptococci (GBS). Although four species of the R antigen have been described for GBS, the R4 proteins is the most prevalent in GBS isolates recovered from humans. This study examined the prevalence of antibodies against the R4 antigen by Western blot (immunoblot) (WB) in sera from 40 mothers colonized with GBS serotype II or III and from 26 monocolonized mothers; 92.5% of the colonized mothers had anti-R4 antibodies, compared with 54% of the noncolonized mothers (P < 0.001). Findings of antibodies in neonatal cord sera (n = 14) were concordant with maternal results by WB analysis for 71% of mother-infant pairs colonized with serotype II and for 57% of pairs colonized with serotype III. Of mothers known to be colonized with type II/R4 or III/R4, 100% (n = 12) had antibody against R4 by WB. This study also evaluated the prevalence of antibody to the GBS R4 antigen in 48 sera from individuals with high and low group A streptococcal anti-DNase B titers. Of those individuals with an anti-DNase B titer of > 640,64% had a positive WB for anti-R4 antibody, compared with 30% of individuals with low anti-DNase B titers (P < 0.05). The R4 antigen of GBS had immunologic identity to the R4 antigen of group A streptococci. Overall, the findings suggested that antibodies to the streptococcal R4 antigen were commonly present in GBS-colonized mothers and that transplacental passage of these antibodies occurred. The presence of antibody to R4 in non-GBS-colonized individuals may be due to immunologic response to past exposure to the R antigen present in GBS or other streptococcal groups.