Uncoupling proteins, members of the mitochondrial carrier family, are present in the mitochondrial inner membrane, and they mediate free-fatty-acid-activated, purine nucleotide-inhibited proton reuptake. Since 1999, it has been shown that uncoupling proteins are present in some eukaryotic microorganisms, including the amoeboid protozoon Acanthamoeba castellanii and the amoeboid slime mold Dictyostelium discoideum. In the mitochondria of these organisms, uncoupling protein activity is revealed by the stimulation of state 4 respiration by free fatty acids accompanied by a decrease in membrane potential. The uncoupling proteins of amoeboid eukaryotes are able to divert energy from oxidative phosphorylation. The functional connection and physiological role of uncoupling protein and alternative oxidase in Acanthamoeba and Dictyostelium species is discussed.