Uncoupling proteins (UCP) found in the inner mitochondrial membrane of mammals dissipate the proton electrochemical gradient across the inner membrane to produce heat rather than synthesize ATP. Using PCR-based methods, we isolated two novel cDNA clones, WhUCP1a and WhUCP1b, that encode the mitochondrial uncoupling protein of wheat (Triticum aestivum L.). The cDNA clones each contain one ORF which can code for a protein of 286 amino acids with a predicted molecular mass of about 30.5 kDa, although three amino acid substitutions are found between them. The deduced amino acid sequences each possess three typical mitochondrial carrier signature domains and six membrane-spanning domains which are highly conserved in the mitochondrial transporter family. Southern analysis suggested that the WhUCP1 gene may be present in as many as three copies in the wheat genome, and also that WhUCP proteins may be encoded by a small multigene family. Northern analysis revealed that the steady-state level of the WhUCP1 mRNA is quite low. Quantitative RT-PCR clearly showed that expression of the WhUCP1 gene in wheat seedlings is insensitive to low temperature. Our data suggest that WhUCP1 might have functions other than low temperature-induced thermogenesis, although WhUCP1 possesses all the typical features reported for known UCPs.