The methanogenic bacterium Methanospirillum hungatei GP1 has been shown to contain two unusual phosphoglycolipids (phosphoglycolipid I and phosphoglycolipid II) that account for 64% (by wt.) of the total cellular lipids. These lipids are derivatives of the dibiphytanyldiglycerol tetraether. One of the free hydroxyls of this tetraether is esterified with glycerophosphoric acid and the other is linked glycosidically to a disaccharide with structure alpha-Glcp-(1 leads to 2)-beta Gal phi in phosphoglycolipid I and beta-Gal phi-(1 leads to 6)-beta Gal phi in phosphoglycolipid II. Smaller amounts of the sn-2,3-diphytanylglycerol analog of phosphatidylglycerol and diglycosyldiphytanylglycerol ethers (DGD-I and DGD-II) containing the same disaccharide residues as in phosphoglycolipid I and phosphoglycolipid II, respectively, were identified, together with very small amounts of diglycosyldibiphytanyldiglycerol tetraethers (DGT-I and DGT-II) containing the same disaccharide residues as in phosphoglycolipid I and phosphoglycolipid II, respectively. A biosynthetic pathway involving head-to-head condensation of phosphatidylglycerol with DGD-I or DGD-II to form phosphoglycolipid I or phosphoglycolipid II, respectively, is proposed.