To elucidate the biosynthesis pathway of linoleic acid and alpha-linolenic acid in Rhodosporidium kratochvilovae YM25235 and investigate the correlation of polyunsaturated fatty acids with its cold adaptation. A 1341 bp cDNA sequence, designated as RKD12, putatively encoding a Delta(12)-desaturase was isolated from YM25235. Sequence analysis indicated that this sequence comprised a complete ORF encoding 446 amino acids of 50.6 kDa. The encoded amino acid sequence shared higher similarity to known fungal Delta(12)-desaturases that are characteristic of three conserved histidine-rich motifs. RKD12 was further transformed into Saccharomyces cerevisiae INVScl for functional characterization. Fatty acid analysis showed the yeast transformants accumulated two new fatty acids: linoleic acid and alpha-linolenic acid. Furthermore, mRNA expression level of RKD12 and the content of linoleic acid and alpha-linolenic acid were increased significantly with the culture temperature downshift from 30 to 15 A degrees C, which might be helpful for the cold adaptation of YM25235. RKD12 is a novel bifunctional a dagger(12)/a dagger(15)-desaturase gene, and the increased RKD12 mRNA expression level and PUFAs content at low temperature might be helpful for the cold adaptation of YM25235.