Uncoupling protein 3 (UCP3) is a muscle mitochondrial protein believed to uncouple the respiratory chain, producing heat and reducing aerobic ATP production. Our aim was to quantify and compare the UCP3 protein levels in type I, IIa and IIx skeletal muscle fibers of endurance-trained (Tr) and healthy untrained (UTr) individuals. UCP3 protein content was quantified using Western blot and immunofluorescence. Skeletal muscle fiber type was determined by both an enzymatic ATPase stain and immunofluorescence. UCP3 protein expression measured in skeletal muscle biopsies was 46% lower ( P=0.01) in the Tr compared to the UTr group. UCP3 protein expression in the different muscle fibers was expressed as follows; IIx>IIa>I in the fibers for both groups ( P<0.0167) but was lower in all fiber types of the Tr when compared to the UTr subjects ( P<0.001). Our results show that training status did not change the skeletal muscle fiber hierarchical UCP3 protein expression in the different fiber types. However, it affected UCP3 content more in type I and type IIa than in the type IIx muscle fibers. We suggest that this decrease may be in relation to the relative improvement in the antioxidant defense systems of the skeletal muscle fibers and that it might, as a consequence, participate in the training induced improvement in mechanical efficiency.