Clinical isolates of group B streptococci from body fluids and mucosal surfaces were examined for production of a trypsin-resistant antigen known as R protein. R protein was extracted with 1% trypsin from cells grown in a semidefined medium. The extracts were tested by immunodiffusion in agarose with a panel of antisera for detection and precise identification of the four species of R protein described by Wilkinson. R antigen was present in 49 of 131 (37%) of the strains tested. Analysis by serotype revealed that 0 of 2 type Ia, 0 of 11 Ib, 1 of 16 (6%) Ia/c, 12 of 15 (80%) II, 0 of 20 II/c, 35 of 49 (71%) III, 0 of 6 IV, and 1 of 12 (8%) nontypeable strains produced R antigen. Production of the R protein and the trypsin-resistant or α component of the c protein appeared to be mutually exclusive. R antigen was more prevalent in isolates from blood (50%) than in those from mucosal sites (27%) for type II strains; no difference was seen for type III strains from these sites. Concordant results were obtained with five paired body fluid-mucosal surface isolates from individual patients and with isolates from 17 mother-baby pairs. The most frequent species of R antigen was R4 (45 of 49), followed by R1 (4 of 49). These two species of R protein were biochemically (trypsin resistant and pepsin sensitive) and immunologically identical to the R-protein antigens produced by prototype strains of groups A, B, and C streptococci.