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Sphingolipids are major components of the plasma membrane, tonoplast, and other endomembranes of plant cells. Previous compositional analyses have focused only on individual sphingolipid classes because of the widely differing polarities of plant sphingolipids. Consequently, the total content of sphingolipid classes in plants has yet to be quantified. In addition, the major polar sphingolipid class in the model plant Arabidopsis thaliana has not been previously determined. In this report, we describe the separation and quantification of sphingolipid classes from A. thaliana leaves using hydrolysis of sphingolipids and high performance liquid chromatography (HPLC) analysis of o-phthaldialdehyde derivatives of the released long-chain bases to monitor the separation steps. An extraction solvent that contained substantial proportions of water was used to solubilized >95% of the sphingolipids from leaves. Neutral and charged sphingolipids were then partitioned by anion exchange solid phase extraction. HPLC analysis of the charged lipid fraction from A. thaliana revealed only one major anionic sphingolipid class, which was identified by mass spectrometry as hexose-hexuronic-inositolphosphoceramide. The neutral sphingolipids were predominantly composed of monohexosylceramide with lesser amounts of ceramides. Extraction and separation of sphingolipids from soybean and tomato showed that, like A. thaliana, the neutral sphingolipids consisted of ceramide and monohexosylceramides; however, the major polar sphingolipid was found to be N-acetyl-hexosamine-hexuronic-inositolphosphoceramide. In extracts from A. thaliana leaves, hexosehexuronic-inositolphosphoceramides, monohexosylceramides, and ceramides accounted for approximately 64, 34, and 2% of the total sphingolipids, respectively, suggesting an important role for the anionic sphingolipids in plant membranes.