Uncoupling protein 3 (UCP3), expressed abundantly in the skeletal muscle, is one of the carrier proteins dissipating the transmitochondrial electrochemical gradient as heat, and thereby has been implicated in the regulation of energy metabolism. We have investigated UCP3 mRNA expression in the widely used L6 myocyte cell line by Northern blot analysis. UCP3 mRNA was not detected in L6 myoblasts, but appeared after their differentiation to myotubes. The UCP3 mRNA level was increased when L6 myotubes were treated with increasing concentrations of triiodothyronine (T3), oleic acid, alpha-bromopalmitate and carbacyclin, a non-selective ligand of peroxisome proliferator-activated receptors (PPARs), whereas it was not influenced when treated with selective ligands of PPARalpha (WY 14¿ omitted¿643) and PPARgamma (troglitazone). A ligand of retinoid X receptor (RXR), 9-cis retinoic acid, was also effective by itself and in combination with carbacyclin in stimulating UCP3 mRNA expression. The mRNA analysis of individual PPAR isoforms revealed that L6 cell expressed a significant level of PPARdelta but undetectable levels of PPARalpha and PPARgamma. These results suggest that UCP3 expression in myocytes is differentiation-dependent and regulated by the T3 receptor, RXR and PPARdelta.