The increasing availability of sequenced genomes enables the reconstruction of the evolutionary history of large protein complexes. Here, we trace the evolution of NADH:ubiquinone oxidoreductase (Complex I), which has increased in size, by so-called supernumary subunits, from 14 subunits in the bacteria to 30 in the plants and algae, 37 in the fungi and 46 in the mammals. Using a combination of pair-wise and profile-based sequence comparisons at the levels of proteins and the DNA of the sequenced eukaryotic genomes, combined with phylogenetic analyses to establish orthology relationships, we were able to (1) trace the origin of six of the supernumerary subunits to the alpha-proteobacterial ancestor of the mitochondria, (2) detect previously unidentified homology relations between subunits from fungi and mammals, (3) detect previously unidentified subunits in the genomes of several species and (4) document several cases of gene duplications among supernumerary subunits in the eukaryotes. One of these, a duplication of N7BM (B17.2), is particularly interesting as it has been lost from genomes that have also lost Complex I proteins, making it a candidate for a Complex I interacting protein. A parsimonious reconstruction of eukaryotic Complex I evolution shows an initial increase in size that predates the separation of plants, fungi and metazoa, followed by a gradual adding and incidental losses of subunits in the various evolutionary lineages. This evolutionary scenario is in contrast to that for Complex I in the prokaryotes, for which the combination of several separate, and previously independently functioning modules into a single complex has been proposed.