The occurrence of a new brassinosteroid of (22S,24R)-3β,22-dihydroxy-5α-ergostan-6-one, named cathasterone, was demonstrated by a GC-MS analysis in cultured cells of Catharanthus roseus. Its endogenous level was in the range of 2–4 ng/g fw, similar to those of brassinolide and castasterone. A feeding experiment with a deuterium-labeled substrate revealed that cathasterone was converted to teasterone and typhasterol. This is the first report of the natural occurrence of cathasterone as a brassinosteroid being the biosynthetic precursor of teasterone.