Iron–sulfur (Fe–S) clusters are essential cofactors in biology due to their use in critical pathways such as nitrogen fixation, photosynthesis, and respiration. Fe–S cluster biogenesis requires complex systems to mobilize iron and sulfide, assemble the nascent cluster, and target Fe–S clusters to downstream target proteins. All of these steps must be coordinated to protect Fe–S cluster biogenesis intermediates from reactive oxygen species and other stressors. Multiple Fe–S cluster biogenesis pathways exist in different organisms and organelles. The Suf ( su lfur f ormation) pathway is phylogenetically the most ancient of the cluster biogenesis systems and is distributed across all domains of life. It can be found as the primary Fe–S cluster biogenesis pathway or as an auxiliary pathway adapted to maintain Fe–S cluster metabolism under stress conditions. Here we describe the biochemical, genetic, and physiological characterization of the Suf system, in all of its iterations.