Two human mAbs (2F5 and 4E10), originally derived from HIV-1-infected patients, are important, but rare, mAbs that exhibit broad cross-clade neutralizing activities against HIV-1. In addition to peptide sequences on the gp41 envelope protein, both antibodies reportedly also bound specifically to several phospholipid antigens. However, the phospholipid binding property of 2F5 has been disputed and, because of uncertainly regarding phospholipid binding, the modeling of neutralizing mechanisms has been difficult. To explore this issue, we examined the binding of 4E10 and 2F5 to a broad range of lipid antigens by ELISA. 4E10 and 2F5 both bound to a variety of purified phospholipids, and 4E10 bound, but 2F5 did not bind, to cardiolipin. Both mAbs also bound to a sulfated glycolipid, sulfogalactosyl ceramide (sulfatide), and to two neutral glycolipids, galactosyl ceramide and glucosyl ceramide, but not to other galactosyl glycolipids. 4E10, but not 2F5, also bound to cholesterol, although both mAbs bound to squalene. Interestingly, 4E10, but not 2F5, exhibited striking binding to lipid A, the lipid moiety of Gram-negative bacterial lipopolysaccharide. The binding properties of 4E10 to phospholipids, sulfatide, cholesterol, squalene, and lipid A were similar to those of a neutralizing murine mAb (WR304) induced by liposomes containing phosphatidylinositol phosphate and lipid A, although WR304 did not bind to neutral glycolipids. The discovery of a binding specificity of 4E10 for lipid A, a widely used vaccine adjuvant, suggests that innate immunity stimulated by lipid A could have played a role for induction of multispecific antibodies that simultaneously recognize both HIV-1 protein and lipid antigens.