Campesterol (3) and dihydrobrassicasterol (4) are biosynthesized from a steroidal 24-ene precursor (desmosterol 1) via 24-methylenecholesterol (2) and 24-methyldesmosterol (5). The stereochemical courses of these transformations have been studied. by feeding regioselectively ^<13>C-labeled steroidal substrates to tissue cultures of higher plants. The formation of 24-methylenecholesterol from desmosterol involves a trans-methylation from S-adenosylmethionine followed by hydrogen migration from C-24 to C-25. The stereochemistry of the hydrogen migration was first investigated. For this purpose, ^<13>C chemical shifts of the diastereotopic C-26 and C-27 methyl groups of 24-methylene-cholesterol were unambiguously assigned by synthesizing the isopropyl pro-R-Me and pro-S-Me ^<13>C-labeled compounds. Feeding of [26-^<13>C]desmosterol to cultured cells of Catharanthus roseus followed by ^<13>C-NMR analysis of the biosynthesized 24-methylenecholesterol established that Re-face hydrogen migration takes place at the C-25 position. Next, chemically synthesized [26,27-^<13>C_2]-24-methyldesmosterol and [26,27-^<13>C_2]-24-methyl-desmosterol were fed to cultures cells of C. roseus and Oryza sativa. The former labeled sterol was shown to be transformed to campesterol and dihydrobrasicasterol, and the latter into sitosterol, demonstrating their intermediary role in the formation of 24-methyl- and 24-ethylcholesterols in higher plants. The stereochemical course of this reduction was explored by feeding regiospecifically ^<13>C labeled compounds, [E-Me-^<13>C]-(5a) and [Z-Me-^<13>C]-(5b) 24-methyldesmosterols. ^<13>C-NMR analysis of the resulting 24-methylcholesterol fraction indicated that the (E)-methyl group turned to be pro-S-Me group of campesterol and pro-R -Me group of dihydrobrassicasterol, thus demonstrating that an anti-mode of hydrogen addition occurs in both cases. This observation implies that the step of the double bond isomerization from 24-methylenecholesterol to 24-methyldesmosterol takes place in such a manner that pro-S-Me group of 2 turns (E)-Me of 5. In conclusion, the present studies established the steric course in the fromation of 24-methyl-cholesterols and allowed to correlate the C-26 and C-27 of the intermediate sterols.