Biotin is a water-soluble vitamin required by all organisms by virtue of its essential role in carboxylation reactions. Although the metabolism and role of biotin in intermediary metabolism are well established, biotin remains one of the most poorly understood water-soluble vitamins in terms of nutritional requirements and responsiveness to physiological and pharmacological states. Significant advances in the understanding of biotin nutriture have been recently accomplished through the description of the kinetics and regulation of biotin transport and improved methods for biotin status assessment. Additionally, the potential role of biotin in the regulation of gene expression has been strengthened through description of altered gene expression during biotin deficiency and through newly described enzymatic activities of the enzyme biotinidase. Given mounting evidence of suboptimum biotin status, a more complete understanding of these aspects of biotin should lead to a greater appreciation of the ways in which biotin aids in the maintenance of health.