We determined the binding of verotoxin-1 (VT1) and verotoxin-2 (VT2) against globotriaosylceramide (Gb3) by a monoclonal antibody (mAb)-based enzyme-linked immunosorbent assay (ELISA). Ethanolic solution of Gb3 containing cholesterol and phosphatidylcholine was passively adsorbed onto the wells of microtiter plate, and Gb3-bound VT1 and VT2 were detected by anti-VT1 and anti-VT2 mAbs, respectively. Although both VT1 and VT2 reacted with Gb3 in a concentration dependent manner, terminal galactose requirement for Gb3 binding was also different from each other. Pretreatment of VT1 showed the inhibitory effect on the binding of VT2 to Gb3, while the VT2-pretreatment showed no inhibitory effect on VT1 binding to Gb3. This was not due to the replacement of Gb3-bound VT2 with post-treated VT1. These results suggest that the binding sites of VT1 and VT2 on Gb3 are not identical to each other.