Glycosphingolipids were extracted from hyphae of Fusarium solani and from an unnamed Fusarium species, and were purified by silica and Iatrobead column chromatography. Their structures were determined by compositional analysis, nuclear magnetic resonance spectroscopy, gas chromatography/mass spectrometry and by fast atom bombardment mass spectrometry of the native and peracetylated materials, which defined their sugar, long-chain base and fatty acid compositions. The locations of the double bonds in the bases were established by 2D NMR spectroscopy and by novel mass spectrometric approaches, including collisional activation of the protonated and lithium-cationized glycosphingolipids, and of the sphingadienene-derived fragment ion at m/z 276. From these results we propose that the structures of the glycosphingolipids from F. solani and Fusarium sp. are N-2'-hydroxyoctadecanoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine and N-2'-hydroxyoctadecenoyl-1-O-beta-D-glucopyranosyl-9-methyl-4, 8-sphingadienine, respectively.