Exposing Arabidopsis (Arabidopsis thaliana) plants to low temperature results in rapid induction of CBF1, 2, and 3 (CBF1-3; also known as DREB1B, C, and A, respectively), which encode transcriptional activators that induce expression of a battery of genes that increase plant freezing and chilling tolerance. Recently, it has been shown that basal levels of CBF3 transcripts and those of certain CBF-regulated genes exhibit circadian cycling. Here, we further explored the regulation of CBF1-3 by the circadian clock. The results indicated that the extent to which CBF1-3 transcripts accumulated in response to low temperature was dependent on the time of day that the plants were exposed to low temperature and that this was regulated by the circadian clock. The highest and lowest levels of cold-induced CBF1-3 transcript accumulation occurred at 4 and 16 h after subjective dawn, respectively. An analysis of CBF2 promoter-reporter gene fusions indicated that this control included transcriptional regulation. In addition, the cold responsiveness of RAV1 and ZAT12, genes that are cold induced in parallel with CBF1-3, was also subject to circadian regulation. However, whereas the maximum level of cold-induced RAV1 transcript accumulation occurred at the same time of day as did CBF1-3 transcripts, that of ZAT12 was in reverse phase, i.e. the highest level of coldinduced ZAT12 transcript accumulation occurred 16 h after subjective dawn. These results indicate that cold-induced expression of CBF1-3, RAV1, and ZAT12 is gated by the circadian clock and suggest that this regulation likely occurs through at least two nonidentical (though potentially overlapping) signaling pathways.Many plants have the ability to sense low temperature and respond by activating mechanisms that lead to an increase in freezing tolerance, an adaptive response known as cold acclimation (Thomashow, 1999;Smallwood and Bowles, 2002). At present, the best understood genetic system that has a role in cold acclimation is the Arabidopsis (Arabidopsis thaliana) CBF coldresponse pathway (Thomashow, 2001). Exposing Arabidopsis plants to low temperature results in rapid induction of a small family of transcriptional activators known either as CBF1, 2, and 3 (CBF1-3; Stockinger et al., 1997;Gilmour et al., 1998;Medina et al., 1999) or as DREB1B, C, and A, respectively (Liu et al., 1998). These transcription factors, which belong to the AP2/ERF domain family of DNA-binding proteins (Riechmann and Meyerowitz, 1998), recognize a cis-acting regulatory element known as the C-repeat/dehydration response element (CRT/DRE; Baker et al., 1994;Yamaguchi-Shinozaki and Shinozaki, 1994;Stockinger et al., 1997) that is present in the promoters of many cold-inducible genes such as COR15A and COR78 (also known as RD29A and LTI78). Transgenic plants overexpressing CBF1, 2, or 3 constitutively express CBFtargeted cold-induced genes, the CBF regulon, and exhibit an increase in freezing tolerance that is independent of a cold stimulus (Jaglo-Ottosen et al., 1998;Liu et al., 1998).Transc...