Taken together, this report gives information about receptor preferences of Stx variants and the role of B-subunits in these receptor interactions. Previous reports suggested that B-subunit activities such as receptor binding and toxin internalization play an important role in determining Stx toxicities. Receptor interaction differences of purified toxoids of Stx1 and Stx2a have been previously reported. However, not much information is available about the receptor interactions of Stx2 variants, which significantly differ in toxicity. Here we report, for the first time, the glycolipid receptor binding preferences of holotoxins and Bsubunits of Stx2 variants. Published KM 11060 studies using thin layer chromatography overlay with Stx B-subunits or high concentrations of Stx holotoxins have demonstrated that Stx2 binds to Gb3 alone, although less effectively than Stx1. In our studies, Stx2a shows strong binding to Gb3 in the presence of PC and Ch. Glycan presentation, or the manner in which the glycans are oriented and displayed to the protein, is known to be a critical factor for binding. It is not clear how glycolipids separated by TLC are oriented. However glycolipids immobilized on a hydrophobic microtiter plate likely replicate the twodimensional display on a biological membrane, where the hydrophobic lipid is attached to the plate and has limited availability compared to the TCN 237 dihydrochloride hydrophilic glycans. Thus we believe the glycolipid immobilized on a hydrophobic microtiter plate in our ELISA studies is more likely to resemble Gb3 presentation in the context of a cellular membrane. The Stx variants displayed distinct glycolipid binding profiles. In most cases the isoforms most toxic to humans, Stx1, Stx2a, Stx2c and Stx2d showed strong glycolipid binding, whereas the weakly toxic form, Stx2b, showed very weak glycolipid binding. This property has diagnostic implications. Capturing Stx by host cell receptors provides a new diagnostic approach to identify and differentiate strains producing Stx variants, which are highly toxic to humans from variants, which are not toxic to humans. The Bmax for Stx1 binding to Gb3 alone was significantly higher than Stx2a binding to Gb3 alone. However the KD values were not very different. It is known that Gb3 binding of Stx2a, but not Stx1, is highly selective. Previous studies demonstrated that cholesterol stabilizes Gb3 in a conformation favorable for binding Stx. It is likely that in the absence of cholesterol, most of the Gb3 does not assume the appropriate conformation to promote binding; however the KD is the same for the few molecule that do assume a conformation favorable for Stx2a binding.