Hairy roots of Ajuga reptans var. atropurpurea produce clerosterol, 22-dehydroclerosterol, and cholesterol as sterol constituents, and 20-hydroxyecdysone, cyasterone, isocyasterone, and 29-norcyasterone as ecdysteroid constituents. To better understand the biosynthesis of these steroidal compounds, we carried out feeding studies of variously 2H- and 13C-labeled sterol substrates with Ajuga hairy roots. In this article, we review our studies in this field. Feeding of labeled desmosterols, 24-methylenecholesterol, and 13C2-acetate established the mechanism of the biosynthesis of the two C29-sterols and a newly accumulated codisterol, including the metabolic correlation of C-26 and C-27 methyl groups. In Ajuga hairy roots, 3alpha-, 4alpha-, and 4beta-hydrogens of cholesterol were all retained at their original positions after conversion into 20-hydroxyecdysone, in contrast to the observations in a fern and an insect. Furthermore, the origin of 5beta-H of 20-hydroxyecdysone was found to be C-6 hydrogen of cholesterol exclusively, which is inconsistent with the results in the fern and the insect. These data strongly support the intermediacy of 7-dehydrocholesterol 5alpha,6alpha-epoxide. Moreover, 7-dehydrocholesterol, 3beta-hydroxy-5beta-cholest-7-en-6-one (5beta-ketol), and 3beta,14alpha-dihydroxy-5beta-cholest-7-en-6-one (5beta-ketodiol) were converted into 20-hydroxyecdysone. Thus, the pathway cholesterol-->7-dehydrocholesterol-->7-dehydrocholesterol 5alpha,6alpha-epoxide-->5beta-ketol-->5beta-k etodiol is proposed for the early stages of 20-hydroxyecdysone biosynthesis. 3beta-Hydroxy-5beta-cholestan-6-one was also incorporated into 20-hydroxyecdysone, suggesting that the introduction of a 7-ene function is not necessarily next to cholesterol. C-25 Hydroxylation during 20-hydroxyecdysone biosynthesis was found to proceed with ca. 70% retention and 30% inversion. Finally, clerosterol was shown to be a precursor of cyasterone and isocyasterone.