A biochemical approach was employed to examine the oxidative utilization of carbohydrate and lipid in red muscle of rainbow trout (Oncorhynchus mykiss) during sustained swimming at 30 and 60% of their critical swimming speed (Ucrit; for 2, 15 and 240 min) and during non-sustainable swimming at 90% Ucrit (for 2, 15 and 45 min). Measurements included pyruvate dehydrogenase (PDH) activity, creatine phosphate, ATP, glycogen, glycolytic intermediates, acetyl-CoA, acetyl-,total-, free-, short-chain fatty acyl- and long-chain fatty acyl- carnitine,intramuscular triacylglycerol and malonyl-CoA concentrations, and whole body oxygen consumption(ṀO2). During the first 2 min at 30 and 60% Ucrit, oxidation of endogenous glycogen by PDH activation increased 4- and 8-fold, respectively, yielding 1.5- to 2.5-fold increases in acetyl-CoA and 2- to 6-fold increases in acetyl-carnitine concentrations. Within 15 min, PDH activity returned to control values (153.9±30.1 nmol g-1 wet tissue min-1); after 240 min there were small 1.7- to 2.6-fold increases in long-chain fatty acyl-carnitine and approx. 50% decreases in malonyl-CoA concentrations, indicating an overall enhancement of lipid oxidation. Sustainable swimming at 30 and 60% Ucrit was further characterized by 1.5- and 2.2-fold increases in ṀO2,respectively. Non-sustainable swimming at 90% Ucrit was characterized by a sustained tenfold (approx.) elevation of red muscle PDH activity (approx. 1600 nmol g-1 wet tissue min-1). Significant 67% decreases in white muscle creatine phosphate and 73% decreases in glycogen levels, without matching increases in lactate levels, point to significant recruitment of white muscle during high-speed swimming for power production, and the potential export of white muscle lactate to red muscle for oxidation. Overall, sustainable exercise at 30 and 60%Ucrit is supported by approximately equal contributions of carbohydrate (approx. 45%) and lipid (approx. 35%) oxidation, whereas non-sustainable swimming is supported primarily by carbohydrate oxidation with only moderate contributions from lipid oxidation.