To explore the role of phospholipid fatty acids in binding of antiphospholipid antibody (aPL) in ELISA, we tested aPL binding to phospholipids containing fatty acids of varying chain length and degree of saturation using direct ELISA and inhibition methods. Polyclonal IgG and IgM human aPL's bind to C18:1 phosphatidylglycerol (PG) better than to C18:0 PG or C18:2 PG. Binding is greater to C18 than to C14:0 or C16:0 PGs; aPL's do not bind to C12:0 PG. aPL binding is not inhibited by C18:1 diacylglycerol, glycerol-3-phosphate, myoinositol, or myoinositol phosphate. The fatty acid chains are critical determinants for antigen recognition and, by projection, biological activity of aPL.