Plants contain a large diversity of sphingolipid structures, arising in part from C4 hydroxylation and Δ4 and Δ8 desaturation of the component long-chain bases (LCBs). Typically, 85–90% of sphingolipid LCBs in Arabidopsis leaves contain acisortransΔ8 double bond produced by sphingoid LCB Δ8 desaturase (SLD). To understand the metabolic and physiological significance of Δ8 unsaturation, studies were performed using mutants of the Arabidopsis SLD genesAtSLD1andAtSLD2. Our studies revealed that both genes are constitutively expressed, the corresponding polypeptides are ER-localized, and expression of these genes inSaccharomyces cerevisiaeyields mixtures ofcis/transΔ8 desaturation products, predominantly astransisomers. Consistent in part with the higher expression ofAtSLD1in Arabidopsis plants,AtSLD1T-DNA mutants showed large reductions in Δ8 unsaturated LCBs in all organs examined, whereasAtSLD2mutants showed little change in LCB unsaturation. Double mutants ofAtSLD1andAtSLD2showed no detectable LCB Δ8 unsaturation. Comprehensive analysis of sphingolipids in rosettes of these mutants revealed a 50% reduction in glucosylceramide levels and a corresponding increase in glycosylinositolphosphoceramides that were restored by complementation with a wild-type copy ofAtSLD1. Doublesld1 sld2mutants lacked apparent growth phenotypes under optimal conditions, but displayed altered responses to certain stresses, including prolonged exposure to low temperatures. These results are consistent with a role for LCB Δ8 unsaturation in selective channeling of ceramides for the synthesis of complex sphingolipids and the physiological performance of Arabidopsis.