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Seed oil bodies are intracellular particles to store lipids as food reserves in oleaginous plants. Description of oil body-associated proteins of Arabidopsis thaliana has been recently reported whereas only few data are available in the case of rapeseed. Oil bodies have been prepared from two double-low varieties of Brassica napus seeds, a standard variety (Explus) and an oleic variety (Cabriolet). Oil bodies have been purified using floatation technique in the successive presence of high salt concentration, detergent or urea in order to remove non-specifically trapped proteins. The integrity of the oil bodies has been verified and their size estimated. Their protein and fatty acid contents have been determined. The proteins composing these organelles were extracted, separated by denaturing gel electrophoresis, digested by trypsin and their peptides were subsequently analyzed by liquid chromatography-tandem mass spectrometry. Protein identification was performed using Arabidopsis thaliana protein sequence database and a collection of Expressed Sequence Tag (EST) of Brassica napus generated from the framework of the French plant genomics programme “Genoplante”. This led to the identification of a limited number of proteins: eight oleosins showing a high similarity each other and representing up to 75% of oil body proteins, a 11 β hydroxysteroid dehydrogenase-like protein highly homologous to the same protein from A. thaliana, and only few contaminating proteins associated with myrosinase activity.