The ecdysteroids from young embryonated eggs of the tobacco hornworm

26-Hydroxyecdysone, which is the major free recoverable ecdysteroid of older age groups of embryonated eggs of the tobacco hornworm was also the major component in 4- to 18-hour-old embryonated eggs. The other 3 beta-ecdysteroids, ecdysone, 20-hydroxyecdysone, and 20,26-dihydroxy-ecdysone, were also present and accounted for all the molting hormone activity; 26-hydroxyecdysone was devoid of molting hormone activity in the house fly assay. 20-Hydroxyecdysone was a minor component, which confirms the earlier observations that the main metabolic route for ecdysteroids during embryonic development is that leading to 26-hydroxyecdysone, whereas formation of 20-hydroxyecdysone is a minor pathway. A new 3 alpha-ecdysteroid, 3-epi-26-hydroxyecdysone, also devoid of molting hormone activity, was the second major ecdysteroid isolated from the eggs. 3-Epi-20,26-dihydroxyecdysone was detected in very minute amounts. In addition to the six 3 beta-and 3 alpha-ecdysteroids there were at least an equivalent number of unknown ecdysteroids all of which lacked molting hormone activity. Their physical properties including chromatographic behavior are discussed.