The binding specificities of the lectins PNA (peanut agglutinin), WGA (wheat germ agglutinin), and UEA I (Ulex europeus agglutinin I) against glycosphingolipids were investigated using an enzyme-linked immunosorbent assay (ELISA), utilizing the biotin-avidin system for detection of bound lectin. PNA showed the highest affinity to GA1, but also bound, though less strongly, to GM1 and GD1b. WGA bound to 3'-nLM1 and 6'-nLM1, the former twice as strongly as the latter, but not to any sialic acid containing glycolipid of the gangliotetraose series. UEA I showed a high affinity for the Lea glycolipid which has an alpha 1-4 linked fucose but not for the glycolipids with alpha 1-3 or alpha 1-2 linked fucose. Interestingly, 3'-nLM1 and nLA1, glycolipids lacking fucose, also bound UEA I. The results show that lectins should be used with caution for establishing terminal sugar sequences in glycosphingolipids.