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Establishing the origin of mitochondria and plastids is key to understand 2 founding events in the origin and early evolution of eukaryotes. Recent advances in the exploration of microbial diversity and in phylogenomics approaches have indicated a deep origin of mitochondria and plastids during the diversification of Alphaproteobacteria and Cyanobacteria, respectively. Here, we strongly support these placements by analyzing the machineries for assembly of iron–sulfur ([Fe–S]) clusters, an essential function in eukaryotic cells that is carried out in mitochondria by the ISC machinery and in plastids by the SUF machinery. We assessed the taxonomic distribution of ISC and SUF in representatives of major eukaryotic supergroups and analyzed the phylogenetic relationships with their prokaryotic homologues. Concatenation datasets of core ISC proteins show an early branching of mitochondria within Alphaproteobacteria, right after the emergence of Magnetococcales. Similar analyses with the SUF machinery place primary plastids as sister to Gloeomargarita within Cyanobacteria. Our results add to the growing evidence of an early emergence of primary organelles and show that the analysis of essential machineries of endosymbiotic origin provide a robust signal to resolve ancient and fundamental steps in eukaryotic evolution.