We analyzed the partition and distribution of a glycolipid on the fracture faces and membrane surfaces of Acanthamoeba castellanii. The glycolipid, a lipophosphoglycan (LPG) with Concanavalin A (Conu A) receptor sites, was labeled with colloidal gold coated with horseradish peroxidase. The label was abundant over exoplasmic faces but absent from protoplasmic faces. Access of lectin and colloidal gold to the interior of lysed cells confirmed the existence of Con A binding sites on the exoplasmic surfaces of vacuoles, but cytoplasmic surface of all cell membranes remained virtually unlabelled. Our results demonstrate the asymmetric topology of glycolipid molecules in a biological membrane. Fracture-label is proposed as a new technique to investigate the distribution and partition of glycolipids in plasma and intracellular membrane halves.