The production of reactive oxygen species (ROS) in mitochondria is very sensitive to the proton motive force and may be decreased by mild uncoupling, mediated e.g. by mitochondrial uncoupling proteins (UCPs). UCPs were conversely hypothesized to be activated by ROS. Conclusions from experiments studying the reactive product of lipid peroxidation 4-hydroxy-2-nonenal (HNE) in isolated mitochondria and UCP knock-out mice are highly controversial. Here we investigated the molecular mechanism of HNE action by evaluating the separate contributions of lipid and protein phases of the membrane and by comparing UCP1 and UCP2, which were reconstituted in planar lipid bilayers. We demonstrated that aldehyde does not directly activate either UCP1 or UCP2. However, HNE strongly potentiated the membrane conductance increase (Gm) mediated by different long-chain fatty acids in UCP-containing and in UCP-free membranes and this suggest the involvement of both lipid-mediated and protein-mediated mechanisms with FA playing the central role. Gm increase was concentration-dependent and exhibited a typical saturation kinetic with the binding constant 0.3 mM. By using Electron Paramagnetic Resonance, membrane fluidity change could be excluded as a cause for the HNE-mediated increase in the presence of FA. The impact of the HNE binding to definite positively charged UCP amino acid residues is discussed as a possible protein-mediated mechanism of the UCP activation.