The terminal enzyme of heme biosynthesis, ferrochelatase (EC 4.99.1.1), catalyzes the insertion of ferrous iron into protoporphyrin IX to form protoheme. Prior to the present work, [2Fe-2S] clusters have been identified and characterized in animal ferrochelatases but not in plant or prokaryotic ferrochelatases. Herein we present evidence that ferrochelatases from the bacteria Caulobacter crescentus and Mycobacterium tuberculosis possess [2Fe-2S] clusters. The enzyme from C. crescentus is a homodimeric, membrane-associated protein while the enzyme from M. tuberculosis is monomeric and soluble. The clusters of the C. crescentus and M. tuberculosis ferrochelatases are ligated by four cysteines but possess ligand spacings that are unlike those of any previously characterized [2Fe-2S] cluster-containing protein, including the ferrochelatase of the yeast Schizosaccharomyces pombe. Thus, the microbial ferrochelatases represent a new group of [2Fe-2S] cluster-containing proteins.