Biotin synthase is responsible for the synthesis of biotin from dethiobiotin and sulfur. Although the name of the protein implies that it functions as an enzyme, it has been consistently reported that biotin synthase produces <1 molecule of biotin per molecule of protein in vitro. Moreover, the source of the biotin sulfur atom has been reported to be the [2Fe-2S] center of the protein. Biotin synthase has therefore been designated as a substrate or reactant rather than an enzyme. We report in vivo experiments demonstrating that biotin synthase is catalytic but that catalysis puts the protein at risk of proteolytic destruction.