The cellular locations of deacylated lipoteichoic acid (dLTA) and lipoteichoic acid (LTA) were examined in late-exponential-phase cells of a serotype III strain of Streptococcus agalactiae (group B streptococci [GBS]) isolated from an infant with late-onset meningitis and compared with a fresh clinical isolate of Streptococcus pyogenes (group A streptococci [GAS]). LTA and dLTA were found to be associated with the protoplast membranes of both organisms, with only dLTA found in mutanolysin cell wall digests. Both organisms released dLTA during growth, but only the GAS released substantial levels of LTA into the culture medium. However, penicillin treatment (5 ,ug/ml for 60 min) of GBS resulted in the recovery of LTA in cell wall digests as well as in the culture medium. These results suggest that under normal growth conditions, the hydrophobic region (glycolipid) of LTA remains associated with the cytoplasmic membrane of GBS and unavailable for hydrophobic interactions at the cell surface with epithelial cells. In contrast, release of LTA into the environment by the GAS allows the fatty acid moieties to interact with hydrophobic domains on the surface of epithelial cells. These results may help explain the marked differences in the specificity of binding between these two major streptococcal pathogens for human fetal and adult epithelial cells.