The influence of D-alanine and carbohydrate substitution of lipoteichoic acids (LTAs) on the binding of antibody directed to the polyglycerol phosphate (PGP) portion was found to be at least partially dependent upon the mode of presentation of the antigen. There were greater differences in binding of anti-PGP immunoglobulins to substituted and unsubstituted LTAs in solution (micellar presentation) than when the same LTAs were adsorbed to the erythrocyte surface, which suggests that there is greater hindrance of access to the PGP chain, possibly as a result of closer packing, in a vesicle or micelle than when LTA is bound to a membrane surface. Although the difference in binding of anti-PGP immunoglobulins was nearly 20-fold between unsubstituted and highly substituted LTAs, rocket heights by rocket immunoelectrophoresis were only 4-fold different for the most highly substituted LTA and unsubstituted LTA. However, unsubstituted LTA clearly bound more immunoglobulin molecules than was reflected in the rocket height alone, since the resulting immunoprecipitates were much more prominent or intense, both before and after staining, than those of highly substituted LTAs. Differences between lightly and moderately substituted LTAs were less than twofold, indicating that under most circumstances estimates of LTA concentrations in samples where the composition is unknown will be within approximately twofold of the estimate.