The respiratory chain alternative enzymes constitute additional pathways to the mitochondrial oxidative phosphorylation (OXPHOS) system, in which oxygen consumption is uncoupled from ATP production. In contrast to the majority of eukaryotic organisms, including other invertebrate animals, vertebrates and arthropods lack any such alternative pathway. However, the expression of alternative oxidases and NADH dehydrogenases from urochordates and fungi in mammalian cultured cells and in the fruitfly Drosophila melanogaster has proven to be benign and to counteract deleterious effects of defective OXPHOS systems, inspiring therapeutic possibilities for mitochondrial and related diseases. The observed benefits are in paradox with the fact that genes for alternative enzymes were lost independently, early in the evolution of both Vertebrata and Arthropoda. I thus propose to investigate the functions of an animal alternative enzyme, the alternative oxidase (AOX), in two research fronts: 1) in studies with Ciona intestinalis (Tunicata: Ascidiacea), that will aim at understanding the biology of this enzyme in its natural “host”; and 2) in studies with transgenic fruitflies expressing the C. intestinalis AOX, which will focus on the possible negative effects caused by this enzyme on the metabolism and fitness of D. melanogaster. I will use a systems biology approach that combines a variety of molecular biology techniques to study general and mitochondrial metabolism under different stress conditions, and assays to test aerobic performance and measure the organism’s fitness, ultimately providing insights into the physiology and the evolution of the alternative pathways in animals.