We cloned the gene and a cDNA for a second CTP: phosphorylcholine cytidylyltransferase (CCT, EC 2.7.7.15) annotated in chromosome 4 by the Arabidopsis genome project, and designated the gene AtCCT2 to discriminate it from the isogene AtCCT1 in chromosome 2. When Arabidopsis plants were chilled at 2 degrees C for 12 h, the level of AtCCT2 transcripts in the rosettes increased about 6-fold over that before 2 degrees C treatment. By contrast, no significant change occurred in the level of AtCCT1 transcripts during 7 d of 2 degrees C treatment. Immunoblot analysis revealed that the level of AtCCT2 in the rosettes chilled at 2 degrees C increased, and that the level of AtCCT1 showed minor changes, when compared with those before cold treatment. Total CCT activity measured at 2 degrees C increased in plants subjected to 2 degrees C treatment, and this increase was sufficient to account for lipid changes induced by the 2 degrees C treatment. We therefore concluded that Arabidopsis utilizes two distinct CCT isozymes for CDP-choline synthesis during cold acclimation. Our findings are important in understanding the physiological functions of CCT isozymes in Arabidopsis and will also stimulate efforts to understand the physiological significance of phosphatidylcholine at low temperatures.