The influence of dietary biotin in horses with brittle hoof horn and chipped hooves was investigated in a long-term study, which was performed over a period from one to six years. 97 horses received 5 mg of biotin per 100 to 150 kg of body weight, per os, daily; 11 horses were not supplemented with biotin and served as controls. The hooves of all horses were evaluated macroscopically every three to four months. Hoof horn specimens of the proximal wall were examined histologically and physically in 25 and 15 horses, respectively. The tensile strength of normal coronary horn was 60 N/mm2 or greater; it was reduced in areas of histological alterations, the lowest value being 20 N/mm2. The hoof horn condition of the biotin-supplemented horses improved after eight to 15 months of supplementation as determined by macroscopic and histologic examinations. The hoof horn condition of most control horses remained constant throughout the study. The growth rate of the coronary horn of horses supplemented with biotin and of control horses was the same. The hoof horn condition deteriorated in 7 of 10 horses after biotin supplementation was reduced or terminated. It was concluded that biotin should be continuously supplemented at the full dosage in horses with severe hoof horn alterations.