The primary aim of the present study was to determine whether intramuscular triacylglycerol (IMTG) utilization contributed significantly to the increase in lipid oxidation during recovery from exercise, as determined from the muscle biopsy technique. In addition, we also examined the regulation of pyruvate dehydrogenase (PDHa) and changes in muscle acetyl units during an 18 h recovery period after glycogen-depleting exercise. Eight endurance-trained males completed an exhaustive bout of exercise (approximately 90 min) on a cycle ergometer followed by ingestion of carbohydrate (CHO)-rich meals (64-70 % of energy from carbohydrate) at 1, 4 and 7 h of recovery. Duplicate muscle biopsies were obtained at exhaustion, and 3, 6 and 18 h of recovery. Despite the large intake of CHO during recovery (491 +/- 28 g or 6.8 +/- 0.3 g kg-1), respiratory exchange ratio values of 0.77 to 0.84 indicated a greater reliance on lipid as an oxidative fuel. However, there was no net IMTG utilization during recovery. IMTG content at exhaustion was 23.5 +/- 3.5 mmol (kg dry wt)-1, and remained constant at 24.6 +/- 2.6, 25.7 +/- 2.8 and 28.4 +/- 3.0 mmol (kg dry wt)-1 after 3, 6 and 18 h of recovery. Muscle glycogen increased significantly from 37 +/- 11 mmol (kg dry wt)-1 at exhaustion, to 165 +/- 13, 250 +/- 18, and 424 +/- 22 mmol (kg dry wt)-1 at 3, 6 and 18 h of recovery, respectively. PDHa was reduced at 6 and 18 h when compared to exhaustion, but did not change during the recovery period. Acetyl-CoA, acetylcarnitine and pyruvate contents declined significantly after 3 h of recovery compared to exhaustion, and thereafter remained unchanged. We conclude that IMTG has a negligible role in contributing to the enhanced fat oxidation during recovery from exhaustive exercise. Despite the elevation of glucose and insulin following high-CHO meals during recovery, CHO oxidation and PDH activation were decreased, supporting the hypothesis that glycogen resynthesis is of high metabolic priority. Plasma fatty acids, very low density lipoprotein triacylglycerols, as well as intramuscular acetylcarnitine stores are likely to be important fuel sources for aerobic energy, particularly during the first few hours of recovery.