Arabidopsis thaliana increases cellular phosphatidylcholine (PC) content during cold acclimation by up-regulating PC biosynthesis. The A. thaliana genes CCT1 and CCT2 encode CTP:phosphorylcholine cytidylyltransferases (CCTs; EC 2.7.7.15), which regulate PC biosynthesis via the CDP-choline pathway. We isolated the T-DNA-tagged knockout mutants cct1 and cct2 of A. thaliana (Wassilevskaja; WS). CCT activity in cct1 and cct2 plants accounted for 29 and 79% to the cellular CCT activity of WS plants, respectively. When plants were exposed to 2 degrees C for 7 d, CCT activity increased in both cct1 and cct2 plants, and immunoblot analyses revealed that cct1 contained an increased level of CCT2 protein whereas cct2 exhibited little increase in CCT1 level. For each mutant grown at 23 degrees C, CCT activity was mainly enriched in the particulate (15,000 x g pellet) and microsomal (150,000 x g pellet) fractions from rosette leaf homogenates. After exposure to cold, the particulate and microsomal fractions of cct1 plants had higher total CCT activity due to increased levels of CCT2; in contrast, the levels of CCT1 in cct2 plants remained unchanged in particulate and microsomal fractions despite a significant increase in the total CCT activity. We conclude that the CDP-choline pathway of A. thaliana is up-regulated at low temperature via isogene-specific modes: enhanced expression of CCT2 and post-translational activation/inactivation of CCT1 in membranes. PC levels were similarly maintained in both mutants and WS plants after 14 d at 2 degrees C, suggesting that either of the CCT genes is sufficient for PC biosynthesis at low temperature.