Globotriaosylceramide [(Gal alpha 1-4Gal beta 1-4Glc-ceramide (Gb3)] was separated from human kidney, and the fatty acid composition was determined. Semisynthetic Gb3 molecular species of corresponding fatty acid chain length were prepared and compared for verotoxin (VT) binding affinity by TLC overlay, and a quantitative binding assay was performed in the presence of auxiliary lipids. Our results indicate that, within the natural range, fatty acid chain length has little effect on verotoxin binding but that Gb3 molecular species containing different fatty acids can interact to provide a higher affinity toxin receptor than any of the individual component receptor species. Receptor function as assayed by TLC overlay was not always found to correlate with binding in a lipid environment. Short-chain fatty acid Gb3 molecular species could not function as VT receptors under these conditions. Evidence is presented to suggest that fatty acid chain length can have a stereoselective effect on carbohydrate conformation.