The role of lipoteichoic acids (LTAs) from Gram-positive bacteria as immunostimulatory molecules was controversial for many years, as inadequate preparation methods as well as heterogeneous and endotoxin-contaminated commercial preparations led to conflicting results. An improved purification methodology for LTA now yields potent bioactive and chemically defined material, which is currently being characterized in various models. A synthetic analogue of Staphylococcus aureus LTA has proven the structure/function relationship. The key role of D-alanine esters for the immune response of LTA was confirmed by synthetic derivatives. The glycolipid anchor of LTA plays a central role analogous to the lipid A of LPS. Methodological aspects and criteria for quality assessment of LTA preparations are discussed.