Strain differences have been postulated to explain the observation that group B Streptococcus type III (GBS III) late-onset disease occurs in only a fraction of colonized infants. To determine the distribution of type-specific polysaccharide antigen (Ag) in GBS III, Ag was measured by rocket immunoelectrophoresis in both supernatant fluids and EDTA extracts and by radial immunodiffusion in multiple HCl extracts of the pellet from cultures of 10 strains of GBS III. Capsular Ag was defined as the sum of Ag in EDTA extracts + Ag in multiple HCl extracts. Both Ag in EDTA extracts and Ag in supernatant fluids correlated with capsular Ag (r = 0.94). GBS III strains were obtained from the blood of 19 infants with late-onset sepsis, from the cerebrospinal fluid or blood of 22 infants with late-onset meningitis, and from mucosal surfaces of both 18 infants and 12 mothers of infants with low levels of type-specific antibody and asymptomatic colonization. Mean values of Ag in supernatant fluids in strains from infants with late-onset sepsis (1.50 +/- 0.08 micrograms/ml) and late-onset meningitis (1.67 +/- 0.09 micrograms/ml) were significantly greater than those in asymptomatic colonization strains (1.14 +/- 0.05 micrograms/ml; P less than 0.001). The number of organisms required for a 50% lethal dose in the chick embryo, determined in 29 strains, was inversely related to Ag in supernatant fluids (r = -0.60). The demonstration that the quantity of capsular Ag produced by GBS III strains is related to their virulence in chick embryos and to their invasiveness in susceptible infants supports the hypothesis that Ag is a virulence factor in humans.