A trial using 12 yearling heifers was carried out to test whether biotin metabolism and bioavailability are influenced by continuous dietary supplementation with biotin. Six of these heifers received no biotin supplementation (controls), while six received a daily dietary supplement of 20 mg biotin over the whole experimental period of four months. During each of three test periods (on days 14 and 21, 56 and 63, and 118 and 124), single test dosages of 40 mg (oral) and 5 mg (intravenous) biotin were given to each animal in a crossover test design. Blood samples were collected up to 72 h after each of these single doses, and at approximately two-weekly intervals for the assessment of baseline values. Serum biotin levels were determined by an ELSA test. Areas under the curves (AUC) were calculated as the target parameter for the assessment of the bioavailability of orally administered biotin. Serum biotin baseline levels were 300-800 ng/l in the controls and 3000-8000 ng/l in the supplemented animals. In both groups, AUC values in the first test period (days 14 and 21) were significantly higher than in subsequent periods. However, the biotin supplementation showed no significant effect. There was no significant difference in elimination half-lives between groups with and without biotin supplementation. The range was 5-18 h. Furthermore, there was no significant difference in the bioavailability of biotin between the test periods or between the biotin-supplemented and unsupplemented animals. Overall bioavailability was 48%.