A macromolecular and adjuvant-active glycolipid lacking the peptide part was separated from wax D preparation of M. tuberculosis strain Aoyama B by silica gel column and preparative thin-layer chromatographies. Although a glycolipid from wax D preparation of M. bovis BCG could also be separated by the same procedure, it was not adjuvant-active. Infrared spectra of both glycolipids were similar but not identical. The main absorptions showed the presence ofhydroxy, methylene and carbonyl groups, and absorption of primary amide and P-O groups in both spectra were very weak or slight. The nitrogen contents of both glycolipids were not detectable (below 0·01%). By alkaline hydrolysis, the glycolipid from M. tuberculosis was separated finally into the hydrophilic and hydrophobic parts forming intermediate products in the process. The molecular weight of the hydrophilic part was around 9300 daltons by gel filtration and it belonged to a fraction of lower molecular weight component(s) in the hydrophilic part of wax D preparation of M. tuberculosis strain H 37Rv.