The distribution of the ganglioside G(M1), at low concentrations, in phase-separated 1:1 dioleoyl phosphatidylcholine (DOPC)/dipalmitoylphosphatidylcholine (DPPC) Langmuir-Blodgett (LB) films transferred on mica was examined by atomic force microscopy (AFM). For concentrations below 1 mol %, G(M1) distributes exclusively in the liquid-condensed DPPC phase where it forms nanometer-scale patches of round and filamentous shapes both at the boundary and toward the middle of DPPC macrodomains. Raising the G(M1) concentration up to 4% increases the number of patches and creates fence like structures at the DOPC/DPPC boundary, whereas some G(M1) clusters might also emerge from the liquid-expanded DOPC phase. Immiscibility of G(M1) with DOPC was confirmed by examination of G(M1)/DOPC LB films. These data strongly support the hypothesis of the existence of G(M1)-enriched domains in biological membranes. They further emphasize the usefulness of the coupling of LB films with AFM detection for the investigation of complex lipid mixtures.