A ganglioside GM1 probe bearing a dark-red fluorescent dye at the sphingosine moiety of the molecule was preparedby a convenient one-pot synthesis. The labeled GM1 permitted the detection of the natural ganglioside GM1 ligandEscherichia coli heat-labile enterotoxin subunit B (EtxB) in picomole quantities on a solid support. When anepitope mapping of several ganglioside binding proteins and protein fragments was performed by screening acellulose membrane-bound synthetic library of 64 16mer peptides with the new probe, several peptides displayingganglioside GM1 affinity could be identified. We consider the labeled glycolipid described herein a versatile toolfor manifold biochemical investigations.