2, 22, 25-Trideoxyecdysone (5β-ketodiol) has been shown to be derived from cholesterol in eggs of the silkworm, Bombyx mori. In order to investigate the difference in ecdysteroid metabolism between diapause eggs and non-diapause eggs of the silkworm, ³H-5β-ketodiol was microinjected into eggs at several stages of early embryogenesis, and the metabolites were characterized using high-performance liquid chromatography. The injected ³H-5β-ketodiol was metabolized not only to free ecdysteroids, but also to conjugates (phosphoric esters of ecdysteroids), in both diapause eggs and non-diapause eggs. Among these metabolites, 20-hydroxyecdysone, which is considered as the active hormone in silkworm eggs, was detected in non-diapause eggs. However, in diapause eggs, various radioactive putative precursors of 20-hydroxyecdysone, such as 2, 22-dideoxyecdysone, 2-deoxyecdysone and ecdysone, were detectable, but ³H-20-hydroxyecdysone was not found. These results suggest that the 20-hydroxylation of ecdysone, which is catalyzed by ecdysone 20-monooxygenase, may be a rate-limiting step in the formation of 20-hydroxyecdysone from ketodiol in the silkworm eggs.