The uncoupling proteins (UCPs) are mitochondrial transporters that modulate the efficiency of oxidative phosphorylation. Members of this family have been described in many phyla within the animal and plant kingdoms, as well as in fungi. The mammalian uncoupling protein UCP1 is activated by fatty acids and inhibited by nucleotides. In the absence of both regulators, UCP1 presents a high ohmic proton conductance that is a unique property of this carrier. The increasing number of protein sequences available has enabled us to apply a sequence analysis approach to investigate transporter function. We reconstructed a robust phylogeny of UCPs and used comparative sequence analysis to search for phylogenetically shared derived sequence features that may confer distinct properties on UCP1. We assessed the functional relevance of shared derived UCP1 residues by substituting them with their counterparts in UCP2, and expressing the protein chimeras in yeast. We found that substitution of both Glu134 and Met140 abolishes the basal proton permeability of UCP1 while preserving fatty acid activation and its nucleotide inhibition.