This study involved a macroscopic evaluation of hoof quality in 152 Lipizzaner horses (130 from Austria and 22 from other countries) and a controlled double blind trial of the effects of biotin on hoof horn growth and quality over 19 months in 42 stallions from the Spanish Riding School (SRS) in Vienna. Using a grading system that incorporated evaluation of horn wall, white line, sole and frog, the macroscopic study revealed the following: 90% of the Austrian Lipizzaners had soft white lines and crumbling, fissured horn at the bearing border of the walls; 39% of the stallions of the SRS, > 4-years-old, had medium to severe hoof horn changes. Daily administration of 20 mg biotin to a test group of horses (n = 26) and a placebo to a control group (n = 16) showed that after 9 months the test group had significantly improved compared to the beginning of the trial and the placebo group (P < 0.01). In the test group, further improvement was observed during the following 5 months and, subsequently, the same good level of hoof condition was maintained over 3 further years of observation. Growth rate of the horn wall was equal in the biotin and placebo group, being 7 mm/28 days, giving a wall renewal period of 11 months. Mean plasma biotin level of untreated horses was 350 ng/l; plasma levels of biotin supplemented horses were > 1000 ng/l. It was concluded that continuous dietary supplementation with biotin at a daily dose of 20 mg is indicated to improve and maintain hoof horn quality in horses with less than optimum quality hoof.