Lipoteichoic acid (LTA) is a component of nearly all gram-positive membranes and recently has been found to be excreted into growth media by certain lactic acid bacteria. Cell-free extracts of LTA are usually contaminated with proteins, polysaccharides, and nucleic acids, thus causing problems to investigators studying the true biological function(s) of LTA. This report describes the preparation of purified extracellular LTA of Streptococcus mutans BHT and intracellular LTA of S. mutans AHT by three techniques: gel filtration, hydrophobic interaction chromatography, and adsorption to phospholipid vesicles. Gel filtration, the most commonly employed method for LTA purification, was found to remove nucleic acids, teichoic acids, and much polysaccharide while greatly concentrating LTA. But gross amounts of antigenic carbohydrate and protein remained associated with the LTA preparation. Hydrophobic interaction chromatography employing octyl Sepharose-4B allowed the separation of protein but not polysaccharide from partially purified BHT LTA preparations. By means of a new technique described in this paper, synthetic membranes (vesicles) were found to effectively separate all contaminants from the intracellular (AHT) and extracellular (BHT) LTA of S. mutans. This rapid method, on a comparative basis, proved to be the most effective approach for the purification of LTA from two widely differing sources.