Transcription factors DREB1A/CBF3 and DREB2A specifically interact with cis-acting dehydration-responsive element/C-repeat (DRE/CRT) involved in cold and drought stress-responsive gene expression in Arabidopsis thaliana. Intact DREB2A expression does not activate downstream genes under normal growth conditions, suggesting that DREB2A requires posttranslational modification for activation, but the activation mechanism has not been clarified. DREB2A domain analysis using Arabidopsis protoplasts identified a transcriptional activation domain between residues 254 and 335, and deletion of a region between residues 136 and 165 transforms DREB2A to a constitutive active form. Overexpression of constitutive active DREB2A resulted in significant drought stress tolerance but only slight freezing tolerance in transgenic Arabidopsis plants. Microarray and RNA gel blot analyses revealed that DREB2A regulates expression of many water stress inducible genes. However, some genes downstream of DREB2A are not downstream of DREB1A, which also recognizes DRE/CRT but functions in cold stress-responsive gene expression. Synthetic green fluorescent protein gave a strong signal in the nucleus under unstressed control conditions when fused to constitutive active DREB2A but only a weak signal when fused to full-length DREB2A. The region between DREB2A residues 136 and 165 plays a role in the stability of this protein in the nucleus, which is important for protein activation.