Cold-induced expression of the Arabidopsis COR (cold-regulated) genes is mediated by a DNA regulatory element termed the CRT (C-repeat)/DRE (dehydration-responsive element). Recently, we identified a transcriptional activator, CBF1, that binds to the CRT/DRE and demonstrated that its overexpression in transgenic Arabidopsis plants at non-acclimating temperatures inducesCORgene expression and increases plant freezing tolerance. Here we report that CBF1 belongs to a small family of closely related proteins which includes CBF2 and CBF3. DNA sequencing of an 8.7 kb region of theArabidopsisgenome along with genetic mapping experiments indicated that the threeCBFgenes are organized in direct repeat on chromosome 4 at 72.8 cM, closely linked to molecular markers PG11 and m600. Like CBF1, both CBF2 and CBF3 activated expression of reporter genes in yeast that contained the CRT/DRE as an upstream activator sequence. The transcript levels for all threeCBFgenes increased within 15 min of transferring plants to low temperature, followed by accumulation ofCORgene transcripts at about 2 h.CBFtranscripts also accumulated rapidly in response to mechanical agitation. The promoter regions of theCBFgenes do not contain the CRT sequence, CCGAC, and overexpression ofCBF1did not have a detectable effect onCBF3transcript levels, suggesting that theCBFgene family is not subject to autoregulation. We propose that cold-induced expression of CRT/DRE-containingCORgenes involves a low temperaturestimulated signalling cascade in whichCBFgene induction is an early event.