Iron-sulfur clusters are universally distributed groups occurring in iron-sulfur proteins. They have a wide range of cellular functions which reflect the chemistry of the clusters. Some clusters are involved in electron transport and energy transduction in photosynthesis and respiration. Others can bind substrates and participate in enzyme catalysis. Regulatory functions have also been documented for clusters that respond to oxygen partial pressure and iron availability. Finally, there are some for which no function has been defined; they may act as stabilizing structures, for example, in enzymes involved in nucleic acid metabolism. The clusters are constructed intracellularly and inserted into proteins, which can then be transported to intracellular targets, in some cases, across membranes. Three different types of iron-sulfur cluster assembly machinery have evolved in prokaryotes: NIF, ISC and SUF. Each system involves a scaffold protein on which the cluster is constructed (encoded by genes nifU, iscU, sufU or sufB) and a cysteine desulfurase (encoded by nifS, iscS or sufS) which provides the sulfide sulfur. In eukaryotic cells, clusters are formed in the mitochondria for the many iron-sulfur proteins in this organelle. The mitochondrial biosynthesis pathway is linked to the cytoplasmic iron-sulfur assembly system (CIA) for the maturation of cytoplasmic and nuclear iron-sulfur proteins. In plant cells, a SUF-type system is used for cluster assembly in the plastids. Many accessory proteins are involved in cluster transfer before insertion into the appropriate sites in Fe-S proteins.