To investigate how the fatty acid composition of membrane lipids influences cell growth and mitochondrial respiration, in particular the expression and capacity of alternative oxidase (AOX), under cold stress, we used the Arabidopsis thaliana fad2 knockout and FAD3+ -overexpressing cultured cells lines affected in extrachloroplastic fatty acid desaturation activities. At 22 degrees C, fad2 mitochondria exhibited a low polyunsaturated fatty acid content and low protein to lipid ratio, while mitochondria from FAD3+ were enriched in linolenic acid and in total membrane protein. As a consequence, both mutants showed a higher membrane microviscosity than the wild type. After exposure to 9 degrees C, FAD3+ mitochondria exhibited lower microviscosity and lower rigidification upon a temperature downshift than fad2. Furthermore, the extent of reduction of cell growth and respiratiory rates in the phosphorylating state was positively related to the cold sensitivity of each cell line, being more pronounced in fad2 that in the wild type, whereas the stability of those parameters reflected the cold resistance of FAD3+. In contrast, an increase in AOX capacity was observed in the three cell lines at 9 degrees C. These inductions were correlated to AOX protein amounts and seem to result from an accumulation of AOX1c transcripts in the three cell lines and of AOX1a transcripts in wild-type and fad2 cells. The fact that there is no direct relationship between the degree of cold tolerance of each cell line and their ability to enhance their AOX capacity suggests that the participation of AOX in the response of Arabidopsis cells to cold stress does not necessarily favor cold tolerance.