An endoplasmic reticulum-localized tomato omega-3 fatty acid desaturase gene (LeFAD3) was isolated. The antisense tomato plants were obtained under the control of the cauliflower mosaic virus 35S promoter (35S-CaMV). Northern blot analysis confirmed that the expression of LeFAD3 was inhibited in the tomato genome. Levels of 18:3 decreased and correspondingly levels of 18:2 increased in total lipids of leaves and roots. After heat stress, the fresh weight of the aerial parts of antisense transgenic plants was higher than that of the wild type (WT) plants. The membrane system ultrastructure of chloroplasts in leaf cells and all of the subcellular organelles in the root tips of transgenic plants remained more intact than those of WT. Relative electric conductivity increased less in transgenic plants than in WT. Under heat stress, the maximal photochemical efficiency of photosystem II (Fv/Fm) and the O(2) evolution rate decreased more in WT than in transgenic plants. These results suggested that the depletion of LeFAD3 increased the saturation of fatty acids and alleviated high temperature stress.