A HvCBF1 cDNA encoding an AP2 domain-containing protein was isolated from barley leaves. Expression of HvCBF1 was induced in barley leaves by exposure to a low temperature (2 degrees C), but not by drought or abscisic acid (ABA) treatment. Functional analysis showed that HvCBF1 was a transcriptional activator, capable of activating expression of reporter genes driven by a cold- and drought-responsive HVA1s promoter in barley leaves. Transactivation analysis of HvCBF1 on a number of synthetic oligonucleotides containing potential C-repeat (CRT)/dehydration-responsive element (DRE) derived from cold-responsive barley genes revealed that HvCBF1 interacted much more efficiently with a GCCGAC motif than a previously identified barley low-temperature-responsive element (LTRE), CCGAAA. A detailed base-substitution mutagenesis study revealed that only the CGAC sequence of the GCCGAC motif was highly conserved for interacting with HvCBF1. The promoter activity of the mutant motifs, ACCGAC and GTCGAC, was 36% and 75% of that of the GCCGAC motif, respectively. The base composition surrounding the GCCGAC motif also had a significant effect on the efficiency of the motif. These data suggest that barley HvCBF1 protein interacts with the (G/a)(C/t)CGAC motif and is involved in regulation of cold-responsive genes in barley via an ABA-independent signal transduction pathway.