Verotoxins bind to glycosphingolipids containing terminal Gal(alpha 1-4)Gal residues. Globotriaosylceramide is the most effective receptor for verotoxin-1 in vitro and is the functional plasma-membrane receptor which mediates cytopathology for most sensitive cells. Binding of verotoxin-1 to a series of galabiose-containing or globotriaose-containing synthetic glycolipids with monoalkylsulfides and bisalkylsulfides or sulfones as the lipid moiety, have been studied for toxin binding by TLC overlay and in solid phase in the presence of auxiliary lipids. The results demonstrate that for an identical carbohydrate, binding is dramatically altered according to the nature of the lipid moiety. The close proximity of the galabiose sequence and the hydrophobic species also compromised recognition. The lipid environment is also a major determinant of receptor function, since species that were effective, even preferred toxin receptors as monitored by TLC overlay, were not necessarily recognized in the presence of auxiliary lipids. Certain glycolipids, which were not recognized by TLC overlay, were nevertheless found to be effective receptors in an auxiliary lipid matrix. These results demonstrate the crucial role of the lipid moiety in verotoxin/glycolipid recognition and are discussed in relation to toxin pathogenesis and glycolipid receptor function.