Localization of fatty acids in biological tissues was made by using TOF-SIMS (time-of-flight secondary ion mass spectrometry). Two cell-types with a specific fatty acid distribution are shown. In rat cerebellum, different distribution patterns of stearic acid (C18:0), palmitic acid (C16:0), and oleic acid (C18:1) were found. Stearic acid signals were observed accumulated in Purkinje cells with high intensities inside the cell, but not in the nucleus region. The signals colocalized with high intensity signals of the phosphocholine head group, indicating origin from phosphatidylcholine or sphingomyelin. In mouse intestine, high palmitic acid signals were found in the secretory crypt cells together with high levels of phosphorylinositol colocalized in the crypt region. Palmitic acid was also seen in the intestinal lumen that contains high amounts of mucine, which is known to be produced in the crypt cells. Linoleic acid signals (C18:2) were low in the crypt region and high in the villus region. Oleic acid signals were seen in the villi and stearic acid signals were ubiquitous with no specific localization in the intestine. We conclude that the results obtained by using imaging TOF-SIMS are consistent with known brain and intestine biochemistry and that the localization of fatty acids is specific in differentiated cells.