The recently discovered UCP3 protein is expressed primarily in muscle and is encoded in a chromosomal region linked to hyperinsulinemia and obesity. Because quiescent skeletal muscle utilizes approximately 45% of whole body oxygen consumption, significant attention has been given to the control and function of UCP3 as a means of regulating energy expenditure and body weight. However, investigation into the regulation of uncoupling protein has been hampered by the inability to assess its activity in vivo. In this study we assess mitochondrial energy uncoupling in skeletal muscle of awake rats by combining [sup. 13] C NMR to measure rates of mitochondrial substrate oxidation with [sup. 31] P NMR to assess unidirectional ATP synthase flux. Male SpragueDawley rats in three groups were used: control (n= 10), 10 day triiodo-L-thyronine ([T. sub. 3]) treated (n= 12), and acute 2, 4-dinitrophenol (DNP) treated (n= 9). UCP3 mRNA and protein levels increased 8.1 [+ or-] 1.1 and 2.8 [+ or-] 0.8 fold respectively in the [T. sub. 3] treated versus control rat gastrocnemius muscle.[sup. 13] C NMR measurements of TCA cycle flux as an index of mitochondrial substrate oxidation were 61 [+ or-] 21, 148 [+ or-] 25, and 310 [+ or-] 48 nmol/g/min in the control,[T. sub. 3], and DNP groups respectively.[sup. 31] P NMR measurements of unidirectional ATPase flux were 83 [+ or-] 14, 84 [+ or-] 14, and 73 [+ or-] 7 nmol/g/s in the control,[T. sub. 3], and DNP groups respectively. Together these flux measurements when normalized to the control group suggest that an acute administration of DNP (mitochondrial uncoupler) and chronic administration of [T. sub. 3] decreases energy coupling by~ 80% and~ 60% respectively and that the latter treatment correlates with an increase in UCP3 mRNA and protein expression. This NMR approach should prove useful for exploring the regulation of uncoupling protein activity in vivo and elucidating its role in energy metabolism and obesity.