Arabidopsis thaliana is frequently used as a model for the study of oilseed biology and metabolism. However, the very small seeds of Arabidopsis can complicate analysis of their oil content and influence the application of results to larger-seeded plants. Here, we describe how seed anatomy, light, and plant-to-plant variation influence the content and measurement of oil in Arabidopsis seeds. The anatomy of Arabidopsis and Brassica napus seeds were compared and the distribution of mass, oil and the fatty acid composition of different seed parts were determined. In Brassica, 90% of the seed oil resides in the cotyledons that contribute 74% of seed mass. By contrast, the values for Arabidopsis are 60% and 45%, respectively, with a higher fraction of the oil deposited in the radicle, hypocotyl, endosperm and seed coat. Growth of Arabidopsis plants with 600 micromol m(-2) s(-1) light resulted in a two-fold higher seed yield, a 40% increase in mass per seed and a 60% increase in oil per seed compared to growth at 100 micromol m(-2) s(-1). Factors that influence the analysis of oil content were evaluated. Intact-seed transmethylation followed by gas chromatography (GC) analysis provided reproducible analysis of Arabidopsis seed oil. However, plant-to-plant variation in oil content is large and we analyzed how this influences the ability to detect statistically valid changes in oil between different genotypes. These observations establish a reference data set on the fatty acid composition and distribution of mass and oil between tissues of Arabidopsis seeds that should help to predict the applicability of results obtained with Arabidopsis to other oilseeds.