A procedure is described for measuring the extraction of lipoteichoic acids from gram-positive bacteria in absolute terms. Virtually complete extraction was achieved from various bacteria by hot phenol/water if the cells were disrupted. Extraction of whole and delipidated cells and of the membrane fraction gave considerably lower yields. Most of the nucleic acids co-extracted from disrupted cells was removed by treatment with nucleases. Nuclease-resistant nucleic acid, protein, polysaccharide, and teichoic acid were separated from lipoteichoic acid by anionexchange chromatography on DEAE-Sephacel or hydrophobic interaction chromatography on octyl-Sepharose. Purified preparations were essentially free of polymeric contaminants, retained their alanine ester substitution, and were in the sodium salt form. Hydrophobic interaction chromatography also made it possible to recognize contamination of lipoteichoic acid with its deacylated and lyso-form, and to discriminate molecular species containing two and three, or two and four acyl groups.