Wheat chromosome 5A plays a key role in cold acclimation and frost tolerance. The major frost tolerance gene Fr-A1(formerly Fr1) and two loci that regulate the transcription of cold- regulated genes (Cor) have previously been mapped on the long arm of this chromosome. In this study we report the discovery of a new locus for frost tolerance designated Fr-A2. This new locus was mapped on the long arm of chromosome 5A of diploid wheat (T. monococcum), 40 cM from the centromere and 30 cM proximal to the major frost tolerance locus Fr-A1. We found also that frost-tolerant and frost-susceptible T. monococcum parental lines differed in the transcription level of the cold induced gene Cor14b when plants were grown at 15 degrees C. Transcription levels of this gene were measured in each of the recombinant inbred lines and mapped as a QTL that perfectly overlapped the QTL for frost survival at the Fr-A2 locus. This result suggested that frost tolerance in this cross was mediated by differential regulation of the expression of the Corgenes. In a previous study in hexaploid wheat (T. aestivum) we had shown that Cor14b was regulated by two loci located on chromosome 5A, one in the same chromosome region as the T. monococcum Fr-A2 locus and the other one closely linked to Fr-A1. Since CBF transcriptional activators in Arabidopsis regulate Corgenes and are involved in frost tolerance, we decided to localize the cold-regulated CBF-like barley gene Cbf3 on the T. monococcum map. This gene was mapped on the peak of the Fr-A2 QTL for frost tolerance. This result suggests that the observed differential regulation of Cor14b at the Fr-A2 locus is due to allelic variation at the XCbf3 locus, and that this transcriptional activator gene might be a candidate gene for the Fr-A2 frost tolerance locus on wheat chromosome 5A.