Lipoglycans were extracted from disrupted cells of Mycobacterium tuberculosis with hot phenol-water. Hydrophobic interaction chromatography on octyl-Sepharose of the crude extract separated nucleic acids and lipoglycans. The latter were retained on the column and fractionated on elution with a propanol gradient into six peaks primarily according to decreasing size of the hydrophilic head groups which dropped from 70 to 5 or 4 monosaccharide residues per molecule. The number of fatty acids per molecule rose from 2.1 to 3.4 over the elution profile, suggesting species containing two, three, and four fatty acids. Peaks I and II and peaks III and IV represented two pairs of lipoarabinomannan and lipomannan, those of peak III and IV containing as a whole smaller hydrophilic head groups and more fatty acids. Peak V and VI are reminiscent of the oligomannosyl derivatives of phosphatidylinositol discovered earlier in mycobacteria. The lipid anchor of all molecular species was shown to be phosphatidylinositol with the extra acyl groups not yet localized. This and an accompanying report complement each other in demonstrating the potential of hydrophobic interaction chromatography for molecular analysis of lipid macroamphiphiles. Lipoteichoic acids, due to continuous small variations in the size of their hydrophilic head groups, separate primarily according to the number of fatty acids, whereas mycobacterial lipoarabinomannans, lipomannans, and phosphatidylinositol mannosides differ as entities so greatly in the size of the hydrophilic headgroup that this quality becomes predominant for separation.