Two independently isolated mutations at the fad7 locus in Arabidopsis produced plants with a temperature-conditional phenotype. Leaves of fad7 mutants grown at 28 degrees C contained less than 30% of wild-type levels of trienoic fatty acids (16:3 plus 18:3) compared with more than 70% of wild-type levels for plants grown at 15 degrees C. Screening of an M(2) population derived from the fad7-1 line led to the identification of a line, SH1, in which the proportion of trienoic acids was much less than in fad7 plants. The segregation pattern of F-2 progeny from a cross between SH1 and wild type indicated that the additional fatty acid mutation in SH1 is at a new locus, designated fads. In a genetic background that was wild type at the FAD7 locus, the fad8 mutation had no detectable effect on overall leaf fatty acid composition irrespective of the temperature at which plants were grown. However, fatty acid analyses of individual leaf lipids revealed small decreases in the levels of 18:3 in two chloroplast lipids. In fad8 plants grown at 22 degrees C, phosphatidylglycerol contained 22.5% 18:3 compared with 33.5% in wildtype Arabidopsis. For sulfoquinovosyldiacylglycerol, the values were 31.4 and 44.5%, respectively. Together with information from studies of the cloned FADE gene (S. Gibson, V. Arondel, K. Iba, C. Somerville [1994] Plant Physiol 106: 1615-1621), these results indicate that the FAD8 locus encodes a chloroplast-localized 16:2/18:2 desaturase that has a substrate specificity similar to the FAD7 gene product but that is induced by low temperature.