The effects of supplementary Biotin on the horn quality of the bovine hoof was studied on 24 dairy cows of different breeds. A comparative trial was set up and the animals were allocated to either a supplemented group (15 animals receiving 10 to 20 mg of Biotin per day) or a control group (nine animals receiving a placebo). The trial lasted for one year during which the herd was visited prior to supplementation and again at regular intervals. Horn and milk samples were taken every four months and the hooves were assessed for clinical status. Horn samples from the heel segment and the abaxial end of the white line were examined by light and transmission electron microscopy, their protein composition was studied by SDS-PAGE and Western Blot. The poor clinical condition of the hind claws at the beginning of the trial improved remarkably in the supplemented group after nine months of additional Biotin and remained unchanged in the control group. Inter- and intracellular changes in the ultrastructure of the horn cell cluster trace back to an effect of Biotin on the energy and lipid metabolism of the differentiating keratinocyte. Changes in ultrastructure and composition of the intercellular cementing substance between the horn cells resulted in an improvement of horn quality. Inside the horn cells the keratin filaments and their associated proteins were more clearly distinguishable compared to the initial status, probably due to changes in the intermolecular linkages. Proteins isolated from bovine hoof horn are separable into two fractions of different electrophoretic mobility. One fraction (14 kD - 30 kD) includes the intermediate filament associated proteins and showed no reaction with the chosen antibodies (CK 10, CK 5/6 and CK 14) against human cytokeratins. The other fraction comprises the keratin proteins with a molecular weight range between 44,5 kD and 57,5 kD. Four acidic and two basic cytokeratins were distinguished by their reaction with two different polyvalent antibodies (AE1 and AE 3) and identified using antibodies against specific human cytokeratins (CK 10, CK 5/6 and CK 14). Postranslational modification of proteins during terminal differentiation reduces the size of the keratinproteins inside the horn cells by about 5 kD compared to the living layers of the epidermis and shows individual variability. Changes in the pattern of protein bands at the end of the trial affected the supplemented group to a greater extent than the control group.