Time-of-flight secondary-ion-mass-spectrometry (TOF-SIMS) was utilized to address the issue of co-localization of cholesterol, phosphocholine and galactosylceramide in rat cerebellar cortex. Rat cerebellum was fixed, freeze-protected by sucrose, frozen and sectioned by cryoultramicrotomy and dried at room temperature. The samples were analyzed in an imaging TOF-SIMS instrument equipped with a Bi(1-7)+-source. The cholesterol signal (m/z 369 and 385) was localized in Purkinje cells and in nuclei of granular layer cells. The phosphocholine headgroup of phosphatidylcholine and sphingomyelin was localized by imaging a specific fragment (m/z 86). This signal was localized in the molecular layer of cerebellar cortex, in Purkinje cells and in parts of the granular layer probably representing the synapse-rich glomeruli. The galactosylceramide was localized by imaging the quasi-molecular ions at m/z 835 and 851, showed a clear colocalization with cholesterol, but also a specific localization in dots (diameter <or=700 nm) in the molecular layer in the vicinity of Purkinje cells, at Purkinje cells and at cells in the granular layer. The results show a heterogeneous distribution of lipids between different cell types not previously described. In order to avoid redistribution artefacts, controls were made by a technique, based on high pressure freezing ,freeze fracturing and freeze drying of samples which were then analyzed by bombardment with a Bi3+ liquid metal ion gun. The galactosylceramide and cholesterol were found distributed as spots in the granular layer. The spots were of homogeneous size with a diameter of <700 nm. Although the galactosylceramide and cholesterol were localized to the same areas, there were clear differences in their distribution at higher resolution.