Cold stress on crops results in severe yield losses through growth retardation and irreversible damage. Recently, Inducer of CBF Expression 1 (ICE1) was identified as the master regulator inducing dehydration responsive element binding protein/C-repeat binding factor (DREB/CBF)-type transcriptional factors involved in the signaling of cold and osmotic stress in Arabidopsis. To examine whether rice ICE homologs function in cold acclimation via regulation of rice DREB homologs in response to cold stress, we assessed a polypeptide epitope containing an ICE-specific motif. Chilling stress on rice seedlings induced two ICE-related proteins with molecular masses of approximately 55 and 40 kDa. These sizes are consistent with those predicted for OsICE1 and OsICE2, respectively. In contrast to the proteins, cold stress had little or no effect on the expression of OsICE1 and OsICE2. Semi-quantitative RT-PCR indicated that both were constantly expressed, but that cold stress sequentially upregulated OsDREB1B, rice heat shock factor A3 (OsHsfA3), and trehalose-6- phosphate phosphatase (OsTPP1). Trehalose treatment enhanced the cold tolerance of seedlings. These results suggest that OsICE homologs function in transcriptional regulation at upstream of a cold-stress-induced transcription factor cascade involving OsDREB1B and OsHsfA3, leading to cold acclimation, possibly involving trehalose synthesis.