Abstract.Immunoblotting under non‐reducing conditions with purified human anti‐Gyaand anti‐Hy locates both antigens to an erythrocyte membrane glycoprotein of apparent Mr46,750–57,500. The antigens are destroyed on intact red cells by the enzymes pronase, trypsin and chymotrypsin, and by treatment with reducing agents. Immunoblotting with anti‐Gyaand anti‐Hy to membranes prepared from red cells pre‐treated with an Endo F preparation caused a mean reduction in apparent Mrof the glycoprotein by 11kDa at the leading and trailing edges, when compared with control membranes. These results suggest that the glycoprotein has one or more complex N‐glycans that are not completely sensitive to Endo F digestion on intact cells. The majority of Gya/Hy‐active molecules are not tightly associated with the red cell membrane skeleton. A gross reduction in reactivity with anti‐Gyaand anti‐Hy by immunoblotting was observed in red cell membranes from patients with paroxysmal nocturnal haemoglobinuria, suggesting a possible membrane linkage via glycosylphosphatidylinositol for the glycoprotein that carries the Gyaand Hy antigens. Immunoprecipitation of the glycoprotein by anti‐Gyashowed that the protein migrates faster under reducing conditions (Mr45,000–54,000). A putative dimer was also evident in the precipitates. The glycoprotein was demonstrated to be distinct from lymphocytefunction‐associated antigen‐3 (CD58), the LWab‐active glycoprotein, the Fya‐active glycoprotein, the Oka‐active glycoprotein a