From the seeds and siliques of Arabidopsis thaliana, six brassinosteroids, brassinolide, castasterone, typhasterol, 6-deoxocastasterone, 6-deoxotyphasterol and 6-deoxoteasterone, were identified by GC-mass spectrometry or GC-selected ion monitoring. As the occurrence of castasterone, typhasterol, 6-deoxocastasterone and 6-deoxotyphasterol in the shoots of A. thaliana has already been reported, this study provides evidence for the occurrence of the above four brassinosteroids in different organs, seeds and siliques, and the first evidence for the occurrence of brassinolide and 6-deoxoteasterone in A. thaliana. All brassinosteroids identified in this study belong to important components of both the early and late C-6 oxidation pathways, which were established in the cultured cells of Catharanthus roseus. This suggests that both pathways are operating in A. thaliana to produce the most biologically active brassinosteroid, brassinolide, which is responsible for growth and development of the plant.