Two new brassinosteroids, (22R,23R,24S)-22,23-dihydroxy-24-methyl-5alpha-cholest-2-en-6-one (secasterol) and (22R,23R,24S)-22,23-dihydroxy-2alpha,3alpha-epoxy-24-methyl-5alpha-cholest-6-one (2,3-diepisecasterone) have been identified together with a known 2,3-epoxybrassinosteroid, secasterone, in seedlings of Secale cereale. Deuterated secasterol, teasterone, and typhasterol, upon administration to rye seedlings, were incorporated into secasterone and 2,3-diepisecasterone, indicating a biosynthetic route via teasterone/typhasterol to secasterol to 2,3-epoxybrassinosteroids.