Mitochondria of amoeba Acanthamoeba castellanii, a non-photosynthetic soil amoeboid protozoon, possess an uncoupling protein (AcUCP) that mediates free fatty acid-activated proton re-uptake dissipating the proton electrochemical gradient built up by respiration. The present study provides the first evidence that UCP could be a cold response protein in unicellulars. In mitochondria isolated from an amoeba batch culture grown temporarily at low temperature (6 degrees C), the content of AcUCP was increased and correlated with an increase in the linoleic acid (LA)-stimulated UCP-mediated carboxyatractyloside-resistant state 4 respiration, as compared to a control culture (routinely grown at 28 degrees C). Moreover, the cytochrome pathway activity was found to be insensitive to the cold exposure of amoeba cells, as indicated by respiration and membrane potential measurements as well as by an absence of change in the adenine nucleotide translocator and cytochrome oxidase expression levels. Furthermore, in mitochondria from the low-temperature-grown cells, at fixed LA concentration, the increased contribution of AcUCP activity to total mitochondrial phosphorylating respiration accompanied by lower coupling parameters was found, as was confirmed by calculation of this contribution using ADP/O measurements.