Cytochrome c (CYC) oxidase (COX), a multisubunit enzyme that functions in mitochondrial aerobic energy production, catalyzes the transfer of electrons from CYC to oxygen and participates in creating the electrochemical gradient used for ATP synthesis. Modeling three-dimensional structural data on COX and CYC reveals that 57 of the >1,500 COX residues can be implicated in binding CYC. Because of the functional importance of the transfer of electrons to oxygen, it might be expected that natural selection would drastically constrain amino acid replacement rates of CYC and COX. Instead, in anthropoid primates, although not in other mammals, CYC and COX show markedly accelerated amino acid replacement rates, with the COX acceleration being much greater at the positions that bind CYC than at those that do not. Specifically, in the anthropoid lineage descending from the last common ancestor of haplorhines (tarsiers and anthropoids) to that of anthropoids (New World monkeys and catarrhines) and that of catarrhines (Old World monkeys and apes, including humans), a minimum of 27 of the 57 COX amino acid residues that bind CYC were replaced, most frequently from electrostatically charged to noncharged residues. Of the COX charge-bearing residues involved in binding CYC, half (11 of 22) have been replaced with uncharged residues. CYC residues that interact with COX residues also frequently changed, but only two of the CYC changes altered charge. We suggest that reducing the electrostatic interaction between COX and CYC was part of the adaptive evolution underlying the emergence of anthropoid primates. mitochondria ͉ electron transport chain ͉ molecular coevolution C ytochrome c (CYC) and the subunits of CYC oxidase (COX) are mitochondrial-functioning proteins that play a central role in aerobic energy production. By catalyzing the transfer of electrons from CYC to oxygen, COX greatly increases an electrochemical gradient used for ATP synthesis. As a consequence of their critical function, mutations altering the structures of either CYC or COX must face the close scrutiny of natural selection. Among vertebrates, relatively few amino acid replacements have occurred in the structures of CYC and COX. However, against this background of slow rates of CYC and COX evolution, upsurges in COX and CYC amino acid replacement rates occurred during the emergence and evolution of the larger brained primates (members of Anthropoidea) (1-12). Here, by using atomic resolution structural data on bovine COX (13) and horse CYC (14) and an experimentally verified model of the interaction between COX and CYC (15, 16), we identify, among the Ͼ1,500 amino acid residues of COX, 57 residues that are likely to bind CYC during delivery of electrons to oxygen. We then demonstrate that the amino acid replacement rate acceleration in anthropoid lineages is especially pronounced in the subset of COX residues that can bind CYC. We further demonstrate that, among these CYC-binding COX residues, many amino acid replacements were from charge-bearing [electros...