The recently discovered phosphoglycolipid, phosphatidylkojibiosyl diglyceride (PKD), was first observed as a biosynthetic by-product of glucosyl diglyceride metabolism inStreptococcus faecalis (faecium) ATCC 9790. Its structure is 1,2-diacyl-3-O(2′-O-α-D-glucopyranosyl-6′-O-phosphoryl-[1″,2″-diacyl-3″-O-sn-glycerol]-α-D-glucopyranosyl)-sn-glycerol. The biosynthesis of phosphatidylkojibiosyl diglyceride occurs by a novel transphosphatidylation reaction in which a phosphatidyl group is transferred from diphosphatidyl glycerol to the primary alcohol function at the 6 position of the internal glucose of kojibiosyl diglyceride. The reaction is catalyzed by a membrane-derived enzyme. Phosphatidylkojibiosyl diglyceride is bound covalently through a phosphodiester bond to the polyglycerol phosphate moiety of membrane lipoteichoic acid fromS. faecalis. Phosphatidylkojibosyl diglyceride has four nonpolar long chain fatty acyl groups and appears to have the necessary physico-chemical properties to anchor the long hydrophilic glycerol phosphate polymer of lipoteichoic acid to the hydrophobic environment of the membrane ofS. faecalis and probably other gram-positive bacteria as well.