The plant hormone abscisic acid (ABA) regulates several physiological and developmental processes in plants, including stress adaptation and seed maturation. ABA-mediated processes appear to be central in plant cold acclimation and expression of cold acclimation-related genes. Ectopic expression of ABI3 encoding a seed-specific transcriptional activator confers on Arabidopsis vegetative tissues the ability to accumulate seed-specific transcripts in response to ABA, and also influences some ABA-mediated vegetative responses. In the present study we characterized the effect of ectopic expression of ABI3 on cold acclimation and development of freezing tolerance in Arabidopsis. We first determined the effect of ABI3 on ABA-induced expression of cold acclimation-related genes. Expression of ABI3 increased the ABA-induced accumulation of transcripts for several ABA/cold/drought-responsive genes such as RAB18 and LTI78. Enhanced expression of these genes was evident even after transient application of ABA, and the enhanced expression was correlated with increased freezing tolerance in ABI3 transgenic plants. Ectopic expression of ABI3 also appeared to modulate low temperature-induced freezing tolerance. The ABI3 transgenic plants acclimated faster than the wild-type plants, and the maximum tolerance obtained was significantly higher. These data showed that lower levels of ABA were needed to trigger the expression of the genes and to maintain the freezing-tolerant state in the ABI3 transgenic plants, and indicate that ectopic expression of ABI3 leads to enhanced responsiveness to ABA. The ectopic expression of ABI3 could provide a new strategy for engineering plant stress tolerance.