Brassinosteroids (BRs) are steroidal plant hormones that are essential for growth and development. It has been proposed that BRs are synthesized via two parallel pathways, the early and late C-6 oxidation pathways according to the C-6 oxidation status. The tomato (Lycopersicon esculentum) Dwarf gene encodes a cytochrome P450 that has been shown to catalyze the C-6 oxidation of 6-deoxocastasterone to castasterone. We isolated an Arabidopsis ortholog (AtBR6ox gene) of the tomato Dwarf gene. The encoded polypeptide has characteristics of P450s and is classified into the CYP85 family. The AtBR6ox and tomato Dwarf gene were expressed in yeast and the ability of the transformed yeast cells to metabolize 6-deoxo-BRs was tested. Metabolites were analyzed by gas chromatography-mass spectrometry. Both enzymes catalyze multiple steps in BR biosynthesis: 6-deoxoteasterone to teasterone, 3-dehydro-6-deoxoteasterone to 3-dehydroteasterone, 6-deoxotyphasterol to typhasterol, and 6-deoxocastasterone to castasterone. Our results indicate that the AtBR6ox gene and the tomato Dwarf gene encode steroid-6-oxidases and that these enzymes have a broad substrate specificity. This suggests that the BR biosynthetic pathway consists of a metabolic grid rather than two separate parallel pathways.