Cell-associated lipoteichoic acids (LTAs) from late-exponential-phase cultures (serotypes Ia, Ib, Ic, II, and III) of group B streptococci isolated from infected and asymptomatically colonized infants were quantitated and characterized by growing the organisms in a chemically defined medium containing [3H]glycerol and [14C]acetate. Cell pellets were extracted with 45% aqueous phenol and chloroform-methanol and subjected to DEAE-Sephacel anion-exchange chromatography. Elution profiles resolved three major peaks, I, II, and III, with glycerol and phosphate present in a 1:1 molar ratio in each peak, and results obtained by Ouchterlony immunodiffusion analysis confirmed the presence of poly(glycerol phosphate). Saponification indicated that [14C]acetate was incorporated into fatty acids of peaks I and II only, suggesting that these were cell-associated LTAs. Peak II was of small molecular weight (less than 10,000) and probably represented another species of LTA. Peaks I and II were further demonstrated to be LTA by their ability to sensitize human type O erythrocytes. Peak III lacked fatty acids and was shown to probably be deacylated LTA. Quantitation of cell-associated teichoic acid material produced by the group B streptococcal strains indicated that the clinical isolates from infants with early- or late-onset disease possessed significantly higher levels than did the asymptomatic (clinical isolates from infants without symptoms of disease) group B streptococcal strains.