[NiFe] hydrogenases catalyze the reversible oxidation of molecular hydrogen into two protons and two electrons. A key organometallic chemistry feature of the NiFe active site is that the iron atom is co-coordinated by two cyanides (CN-) and one carbon monoxide (CO) ligand. Biosynthesis of the NiFe(CN)(2)(CO) cofactor requires the activity of at least six maturation proteins, designated HypA-F. An additional maturase, HypX, is required for CO ligand synthesis under aerobic conditions, and preliminary in vivo data indicated that HypX releases CO using N-10-formyltetrahydrofolate (N-10-formyl-THF) as the substrate. HypX has a bipartite structure composed of an N-terminal module similar to N-10-formyl-THF transferases and a C-terminal module homologous to enoyl-CoA hydratases/isomerases. This composition suggested that CO production takes place in two consecutive reactions. Here, we present in vitro evidence that purified HypX first transfers the formyl group of N-10-formyl-THF to produce formyl-coenzyme A (formyl-CoA) as a central reaction intermediate. In a second step, formyl-CoA is decarbonylated, resulting in free CoA and carbon monoxide. Purified HypX proved to be metal-free, which makes it a unique catalyst among the group of CO-releasing enzymes.