AbstractFollowing injection into femaleManduca sextapupae, [14C]cholesterol is converted to a radiolabeled C21nonecdysteroid conjugate as well as ecdysteroid conjugates, which in ovaries and newly‐laid eggs consist mainly of labeled 26‐hydroxyecdysone 26‐phosphate. During embryogenesis, as the level of 26‐hydroxyecdysone 26‐phosphate decreases there is a concurrent increase in the amount of a new, labeled ecdysteroid conjugate. This conjugate, which is the major ecdysteroid conjugate (9.4 μg/g) in 0‐ to 1‐hour‐old larvae was identified as 26‐hydroxyecdysone 22‐glucoside by nuclear magnetic resonance and chemical ionization mass spectrometry. This is the first ecdysteroid glucoside to be identified from an insect. The disappearance of 26‐hydroxyecdysone 26‐phosphate in 0‐ to 1‐hour‐old larvae indicates that the 26‐hydroxyecdysone 22‐glucoside is derived from 26‐hydroxyecdysone 26‐phosphate. 3‐Epi‐26‐hydroxyecdysone was the major free ecdysteroid isolated from these larvae and 3‐epi‐20,26‐dihydroxyecdysone was the next most abundant ecdysteroid isolated. Interestingly, the 0‐ to 1‐hour‐old larvae contained the highest levels of 3α‐ecdysteroids per gram of insect tissue (8.7 μg/g) to be isolated from an insect, yet there was a complete absence of the corresponding free 3β‐epimers. The ecdysteroid conjugate profiles of ovaries and 0‐ to 1‐hour‐old larvae are discussed. Methodology is presented that permits the efficient separation of free and conjugate