Human and rodent uncoupling protein (UCP)3 mRNA is upregulated after acute exercise. Moreover, exercise increases plasma levels of free fatty acid (FFA), which are also known to upregulate UCP3. We investigated whether the upregulation of UCP3 after exercise is an effect of exercise per se or an effect of FFA levels or substrate oxidation. Seven healthy untrained men [age: 22.7 +/- 0.6 yr; body mass index: 23.8 +/- 1.0 kg/m(2); maximal O2 uptake (VO2 max): 3,852 +/- 211 ml/min] exercised at 50% VO2 max for 2 h and then rested for 4 h. Muscle biopsies and blood samples were taken before and immediately after 2 h of exercise and 1 and 4 h in the postexercise period. To modulate plasma FFA levels and fat/glucose oxidation, the experiment was performed two times, one time with glucose ingestion and one time while fasting. UCP3 mRNA and UCP3 protein were determined by RT-competitive PCR and Western blot. In the fasted state, plasma FFA levels significantly increased (P < 0.0001) during exercise (293 +/- 25 vs. 1,050 +/- 127 micromol/l), whereas they were unchanged after glucose ingestion (335 +/- 54 vs. 392 +/- 74 micromol/l). Also, fat oxidation was higher after fasting (P < 0.05), whereas glucose oxidation was higher after glucose ingestion (P < 0.05). In the fasted state, UCP3L mRNA expression was increased significantly (P < 0.05) 4 h after exercise (4.6 +/- 1.2 vs. 9.6 +/- 3.3 amol/microg RNA). This increase in UCP3L mRNA expression was prevented by glucose ingestion. Acute exercise had no effect on UCP3 protein levels. In conclusion, we found that acute exercise had no direct effect on UCP3 mRNA expression. Abolishing the commonly observed increase in plasma FFA levels and/or fatty acid oxidation during and after exercise prevents the upregulation of UCP3 after acute exercise. Therefore, the previously observed increase in UCP3 expression appears to be an effect of prolonged elevation of plasma FFA levels and/or increased fatty acid oxidation.