The CBF/DREB1 transcription factors control an important pathway for increased freezing and drought tolerance in plants. We report here the isolation of one CBF/DREB1-like gene, CBF4, from both freezing-tolerant wild grape (Vitis riparia) and freezing-sensitive cultivated grape (Vitis vinifera). The deduced protein in V. riparia is 99% identical to the corresponding protein in V. vinifera; 45-48% to three other Vitis CBF proteins reported earlier and 57% to AtCBF1, and contains CBF-specific amino acid motifs. Agroinfiltration experiments in tobacco leaves revealed that VrCBF4 activates expression from reporter genes driven by a CRT-containing promoter. Expression of the endogenous Vitis CBF4 genes was low at ambient temperature, but enhanced upon exposure to low temperature (4 degrees C). Uncommon for CBF genes, this expression was maintained for several days. No significant difference in expression pattern was observed between V. riparia and V. vinifera. Vitis CBF4 was expressed in both young and mature tissue, in contrast to the previously described Vitis CBF1, 2 and 3. Together, these results suggest that CBF4 represents a second type of CBF in grape that might be more important for the over-wintering of grape plants.