CBF͞DREB1 (C-repeat-binding factor͞dehydration responsive element-binding factor 1) genes encode a small family of transcriptional activators that have been described as playing an important role in freezing tolerance and cold acclimation in Arabidopsis. To specify this role, we used a reverse genetic approach and identified a mutant, cbf2, in which the CBF2͞DREB1C gene was disrupted. Here, we show that cbf2 plants have higher capacity to tolerate freezing than WT ones before and after cold acclimation and are more tolerant to dehydration and salt stress. All these phenotypes correlate with a stronger and more sustained expression of CBF͞DREB1-regulated genes, which results from an increased expression of CBF1͞DREB1B and CBF3͞DREB1A in the mutant. In addition, we show that the expression of CBF1͞DREB1B and CBF3͞DREB1A in response to low temperature precedes that of CBF2͞DREB1C. These results indicate that CBF2͞DREB1C negatively regulates CBF1͞DREB1B and CBF3͞ DREB1A, ensuring that their expression is transient and tightly controlled, which, in turn, guarantees the proper induction of downstream genes and the accurate development of Arabidopsis tolerance to freezing and related stresses.F reezing temperatures greatly limit the geographical distribution and growing season of plants and cause negative effects on crop quality and productivity. As a consequence, appreciable effort has been conducted to determine the adaptive mechanisms plants have evolved to survive this adverse environmental condition. Many plants, including Arabidopsis, increase their freezing tolerance in response to low, nonfreezing temperatures. This process, called cold acclimation (1), is complex and involves a number of biochemical and physiological changes, ranging from alterations in lipid composition to accumulation of sugars (2). Different studies have suggested that low-temperature-regulated gene expression is critical in plants for cold acclimation (2). Low-temperature-responsive genes encode a diverse number of proteins, including enzymes involved in respiration and metabolism of carbohydrates, lipids, phenylpropanoids and antioxidants, molecular chaperones, antifreeze proteins, among others, with a believed function in freezing tolerance (2).During the past few years, substantial progress has been made toward understanding how low temperatures regulate gene expression. In particular, a family of transcription factors in Arabidopsis known either as C-repeat-binding factor (CBF)1, CBF2, and CBF3 (3, 4) or dehydration-responsive elementbinding factor (DREB)1B, DREB1C, and DREB1A (5), respectively, has been identified. These factors belong to the Apetala 2͞ethylene-responsive element-binding protein (AP2͞ EREBP) family of DNA-binding proteins (6) and bind to the cold-and dehydration-responsive DNA regulatory element (DRE) (7), also termed C-repeat (CRT) (8). CRT͞DRE elements contain the conserved CCGAC core sequence, which is sufficient to induce gene transcription under cold stress (7,8) and is present in the promoters of many cold-induc...