Our previous data showed that aldehydic lipid peroxidation products, interacting with mitochondrial membrane lipids, could alter the physicochemical status of the membrane. This study was initiated to examine the interaction of these aldehydes with a major mitochondrial protein, the adenine nucleotide translocator (ANT). Our findings showed that the transporting activity of ANT in intact mitochondria was inhibited by two unsaturated aldehydes, 4-hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE). To probe further into the underlying mechanism of this inhibition, a reconstituted ANT model was developed by incorporating isolated ANT into liposomes. Pretreatment of ANT with HNE prior to reconstitution resulted in decreased activity in the reconstituted ANT. Further investigation revealed that this decreased activity was probably due to loss of sulfhydryl groups, which are essential for ANT activity. Interestingly, pretreatment of the liposomes with HNE also caused a decrease in the reconstituted ANT activity by indirectly altering the physiochemical status of the lipid environment in which ANT was embedded. These results demonstrate that the reactive aldehydes derived from mitochondrial lipid peroxidation can impair the membrane function by interacting with both the protein and the lipid moieties in the membrane. Thus, the varied damaging effects associated with lipid peroxidation may be mediated by their secondary aldehydic byproducts.