1. The mechanism of formation of the A/B cis ring junction of ecdysteroids in the locust Schistocerca gregaria, was investigated by incorporation of [4-14C,3 alpha-3H], [4-14C,4 alpha-3H] and [4-14C,4 beta-3H]cholesterol into 20-hydroxyecdysone in fifth-instar larvae and into ecdysteroid conjugates in ovaries of maturing adult females. 2. In both systems there was retention of the 4 alpha-3H atom in the ecdysteroid and elimination of the 3 alpha- and 4 beta-3H atoms. 3. The 3H retained in the ecdysone formed from [4 alpha-3H]cholesterol in the ovarian system was probably located at C-4. The results are interpreted by postulating the involvement of a 3-oxo-delta 4 intermediate in ecdysteroid biosynthesis in insects.
