Biotin is biosynthesized in four steps from pimeloyl CoA. KAPA synthase which catalyzes the condensation fo pimeloyl CoA with alanine has been purified, kinetically characterized and its mechanism is discussed. The insertion of sulfur into dethiobiotin could be studied only in vivo. Experiments with different precursors indicate that the first step is very likely the introduction of sulfur on the methyl group, followed by cyclization.