An investigation was undertaken to clarify the biochemical composition of the equine hoof, and to obtain basic data which will serve for clarification of the relationship between the biochemical composition of the hoof and the diseases of the laminae. Little information has been available on lipids contained in the hoof. It is presumed that the lipid in the hoof may be related to the permeation of water or the hardness of the hoof. In this investigation, the protein content, lipid constituent, and ash in the fore and hind hoofs of two healthy Thoroughbred horses were determined. The results obtained are as follows: (1) The coefficient of nitrogen for hoof protein was 5.93, which was lower than that of general protein. It seems to be useful for the study of equine hoofs in future. (2) Lipid was a less important component of the hoof than protein. Of the lipid constituents, sterols, triglycerides, and sterolesters in the nonpolar lipids and ceramide dihexoside in the polar lipids were identified by silica-gel column and thin-layer chromatography. (3) Palmitic, stearic, and oleic acids of the main fatty acids in the nonpolar lipids and lignoceric acid of those in the polar lipids, including the former three acids, were detected by gas chromatography. (4) From the data mentioned above, it was suggested that the lipid should be taken into consideration for the protection of the equine hoof.