Hydrophobic interaction chromatography fractionated the lipoteichoic acid of Enterococcus faecalis into species of decreasing poly(glycerophosphate) chain length and decreasing extent of substitution with alpha-kojibiosyl residues (Glcp alpha 1----2Glcp alpha 1----). The chain length varied between 14 and 33 glycerophosphate residues per lipid anchor, the extent of glycosylation between 0.18 and 0.44 mol of alpha-kojibiosyl residues per mole of phosphorus, and, accordingly, the number of alpha-kojibiosyl substituents per chain between 3 and 15. Almost identical values were obtained when the same lipoteichoic acid was chromatographed on DEAE-Sephadex and concanavalin A, which separate molecular species according to increasing number of phosphate groups and alpha-kojibiosyl residues, respectively. Species from all three columns, which were identical in chain length and glycosylation, also had similar fatty acid patterns. These results prove the suitability of all three procedures for species analysis. One advantage of hydrophobic interaction chromatography over the other two procedures lies in its broader applicability since it is not dependent on negative charges or specifically binding oligosaccharide structures. Another advantage is the capacity of hydrophobic interaction chromatography to separate molecular species differing in the number of fatty acids [W. Fischer, H.U. Koch, and R. Haas (1983) Eur. J. Biochem. 133, 523-530] and render them accessible to molecular analyses.