Many plants living in temperate regions acquire freezing tolerance after exposure to non-freezing low temperatures over a certain period. This phenomenon is known as cold acclimation. To search novel genes involved in cold acclimation, mutants of Arabidopsis thaliana that have an increased freezing tolerance without cold acclimation were isolated using the T-DNA activation-tagging method. Calli derived from hypocotyls of about 33,000 seedlings were co-cultivated with Agrobacterium tumefaciens harboring pPCVICEn4HPT that contains enhancers originated from the 35S promoter of cauliflower mosaic virus, to generate the activation-tagging callus lines. The T-DNA-inserted calli were selected for their ability to grow in the callus-inducing medium (CIM) containing hygromycin for two weeks and to remain alive after a subsequent freezing treatment at -14℃ for 3 h. Consequently, only 47 transformant lines were found to survive. In 34 of these lines, loci of insertions of T-DNA were determined by thermal asymmetric interlaced-PCR (TAIL-PCR): single T-DNA insertions were seen in 28 lines, while double insertions in 6 lines. Furthermore, we harvested seeds from regenerated plants of these tagging lines and re-evaluated freezing tolerance of the calli from the seedlings. The calli from 6 mutant lines were more freezing-tolerant than that from the wild-type plants. In two of these mutant lines examined with semi-quantitative reverse transcription-PCR (RT-PCR), a gene located near the insertion of enhancer elements was activated. A basic local alignment search tool (BLAST) search showed that the gene activated in the one line encodes a protein with an Armadillo-like helical motif and that activated in the other line encodes a protein belonging to a fibrillin family