Administration of β-adrenergic receptor (β-AR) agonists, especially β₃-AR agonists, is well known to increase thermogenesis in rodents and humans. In this work we studied the role of the β₃-AR in regulating mRNA expression of genes involved in thermogenesis, i.e., mitochondrial uncoupling proteins UCP2 and UCP3, and peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1), in mouse skeletal muscle. For this purpose, different β₃-AR agonists were administered acutely to both wild type mice and mice whose β₃-AR gene has been disrupted (β₃-AR KO mice). CL 316243 increased the expression of UCP2, UCP3 and PGC-1 in wild type mice only. By contrast, BRL 37344 and CGP 12177 increased the expression of UCP2 and UCP3 in both wild type and β₃-AR KO mice, whereas they increased the expression of PGC-1 in wild type mice only. Finally, acute (3 h) cold exposure increased the expression of UCP2 and UCP3, but not PGC-1, in skeletal muscle of both wild type and β₃-AR KO mice. These results show that selective stimulation of the β₃-AR affects the expression of UCP2, UCP3 and PGC-1 in skeletal muscle. This effect is probably indirect, as muscle does not seem to express β₃-AR. In addition, our data suggest that BRL 37344 and CGP 12177 act, in part, through an as yet unidentified receptor, possibly a β₄-AR.