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The lipids of horse hoof have been analyzed by quantitative thin-layer chromatography. The major components include cholesterol (37-40%), six groups of ceramides (10-15%), and cholesteryl sulfate (15-20%). Free fatty acids are abundant (15.8%) in the outer fully keratinized hoof, but are present at only low levels (3.1%) in the softer hyponychium. The material identified as cholesteryl sulfate was isolated by preparative thin-layer chromatography and characterized by a combination of chemical, chromatographic, and spectroscopic methods. The infrared spectrum of the isolated material had absorption bands at 800, 1063, 1200, and 1235 cm-1, indicating a sulfate ester. This sulfolipid was nonsaponifiable, but upon acid hydrolysis yielded cholesterol as the only charrable product, which was identified by its chromatographic behavior and by its electron impact mass spectrum. The isolated sulfolipid also had the same mobility on thin-layer chromatography as authentic cholesteryl sulfate in several different solvent systems. Sulfated gangliosides, which were previously reported to be major horse hoof lipids, were not found among the principal lipid components in the present study. It is concluded that cholesteryl sulfate is the major polar lipid of horse hoof. This may be a significant factor determining the high degree of cohesiveness of this fully keratinized tissue.