The overall fatty compositions of leaf and root lipids from a mutant of Arabidopsis thaliana were characterized by reduced levels of linolenate (18:3) and correspondingly elevated levels of linoleate (18:2) as a consequence of a single nuclear mutation at the fad3 locus. Comparison of the fatty acid compositions of individual lipids from wild type and mutant plants showed that chloroplast lipids were largely unaffected by the mutation, whereas each of the phospholipids synthesized on the endoplasmic reticulum in the mutant exhibited a marked reduction in the proportion of 18:3 relative to wild type. These and other results indicate that the fad3 mutants are deficient in the activity of an endoplasmic reticulum 18:2 desaturase. In root tissues, this enzyme appears to account for over 80% of the 18:3 present. However, in young leaves of the mutant, phosphatidylcholine (the major phospholipid) contains 16% 18:3 compared with 29% in the wild type, and the proportion of 18:3 in this lipid increases with plant age in both wild type and mutant plants. These results reflect an exchange of lipid between the chloroplast and endoplasmic reticulum that allows the chloroplast desaturases to provide highly unsaturated lipids to the extrachloroplast membranes of leaf cells.