The central part of cold-acclimated rosettes of Arabidopsis thaliana L. (ecotype Columbia) survived freezing at lower temperatures better than did those at the rosette periphery. Electrolyte-leakage tests with detached leaves verified that freezing tolerance in central (or young) leaves increased faster and to a greater extent than in peripheral (or aged and mature) leaves at 2 degrees C. Cold-induced accumulation of sugars could partly account for the leaf-order-dependent enhancement of freezing tolerance after 1 d at 2 degrees C, whereas the role of proline remains to be determined. Cold-induced accumulation of the transcripts of stress-inducible CBF/DREB1 transcription factors apparently disagreed with the observed difference in the freezing tolerance in different leaf orders. However, the levels of COR78/RD29A transcripts were almost the same between different leaf orders after 1-3 d at 2 degrees C, and COR78/RD29A content per total leaf protein was similar between different leaf orders after 7 d at 2 degrees C. Thus, cold-induced accumulation of COR78/RD29A does not seem to account for the observed difference in freezing tolerance in different leaf orders. Although further studies are required for comprehensive understanding of the phenomenon, the present work does provide an important and interesting physiological aspect in our understanding of the freezing tolerance in plants.