Uncoupling protein-3 (UCP3), a mitochondrial membrane transporter, is a candidate effector of thermogenesis. Even though mice with targeted disruption of the UCP3 gene are not obese, indirect evidence suggests that this protein contributes to the control of energy expenditure in humans. We therefore characterized the human UCP3 gene and compared it with its rodent homologues with respect to tissue-specific expression and regulatory regions. Like rodent UCP3, human UCP3 was expressed in skeletal muscle and brown adipose tissue (BAT). The short mRNA isoform, UCP3(S), which is absent in rodents, was relatively more abundant in human skeletal muscle in comparison to human BAT. Two tissue-specific transcription start sites for each skeletal muscle and BAT were delineated for human UCP3. Tissue-specific transcript initiation was maintained in both tissues and cultured cells over a wide range of expression levels. In contrast, rodent transcripts were initiated at the same site in BAT and muscle tissue. Comparison of human and rodent promoters indicated a rapid phylogenetic evolution suggesting functional diversification. The transcription from tissue-specific promoters in humans is a novel finding that may provide the basis for therapeutic interventions aimed at regulating energy expenditure in a tissue-specific fashion.