Using a specially designed, motorised t.l.c.-f.a.b.-m.s. probe with continuous desorption and scanning over a moving t.l.c. plate, it was shown that glycolipids with identical carbohydrate sequences were well resolved into molecular species with differences in long-chain base and fatty acid. There was no serious diffusion of the glycolipids into the matrix. The technique is demonstrated for sulphatides (one and two sugar residues) isolated from human kidney, GM3 ganglioside isolated from human malignant melanoma, and chemically modified gangliotetraosylceramide from mouse intestine. T.l.c.-f.a.b.m.s. is convenient for sequencing and composition analysis of receptor-active glycolipids, the biological activity of which can be monitored in parallel by overlay on the t.l.c. plate with proteins, viruses, bacteria, or animal cells.