Biotin-dependent carboxylases catalyze a variety of carboxyl transfer reactions in a number of metabolic pathways and are found in all free-living organisms. They are large molecules which can comprise a single polypeptide chain with three domains or up to three subunits, each of which performs a particular part of the overall reaction. Biotin plays a central role in the action of these enzymes. In this Account we examine the current state of knowledge of the chemistry of catalysis and consider how the recent explosion of available protein sequence and structural information has assisted our understanding of the mechanisms of biotin-dependent enzymes.