The fate of all the carbons and hydrogens originating from MVA were revealed by 13C NMR spectroscopy in the biosynthesis of phytosterols. The 1,2-methyl migrations (14β→13β and 8α→14α) were demonstrated in the formation of cycloartenol (2) in Physalis peruviana callus fed with [1,2-^<13>C_2]acetate. Also the 1,2-hydride shifts (17β→20, 13α→17α, 9β→8β, and 24→25) were verified in 24-methylenecycloartanol (5) biosynthesized from [2-^<13>CD_3]acetate in Trichosanthes kirilowii var. japonica callus by observing β-deuterium isotopically shifted signals. The both C-11 and C-12 of sitosterol (10) biosynthesized in R. japonica callus in the presence of [5-^<13>CD_2]MVA were labeled in two ways, ^<13>CD_2 and ^<13>CDH, showing that squalene is released from the enzyme involved. The following oxidation reaction does not distinguish one farnesyl moiety from the other when forming squaleneoxide (9A) and (9B). In 24α-ethylsterol (6), no deuterium atom around C-24 was observed and the hydride shift (24→25) was detected in the 24-methylene derivative (5). However, in the case of 24β-ethyl-sterols (24) and (26) obtained from T. kirilowii fed with [2-^<13>CD_3]acetate, the C-24 was found to bear a deuterium atom. The C-26 of (25) and C-27 (pro-S) of (27) were observed as doublets labelled from [1,2-^<13>C_2] acetate. These findings suggest that the Δ^<25> double bond in (24) is formed with a hydrogen elimination from the methyl group at C-25 originating from C-6 of MVA, then hydrogen attack on the 25-re-face of the double bond occurs to form (26). The C-27 of this final product is from the C-6 of MVA.