1. [7α- 3 H 1 ]- and [7β- 3 H 1 ]-Cholesterol were synthesized by a modified method. 2. The stereochemistry of Δ 7 -bond formation during ecdysone and ecdysterone biosynthesis in the insect, Calliphora erythrocephala and the plants, Taxus baccata and Polypodium vulgare was investigated by using [4- 14 C,7α- 3 H 1 ]cholesterol and [4- 14 C,7β- 3 H 1 ]cholesterol. 3. In each case, the 7β hydrogen was stereospecifically eliminated. 4. The possible significance of the results is discussed in relation to double-bond formation in other systems and the stage at which the Δ 7 bond is introduced during ecdysone biosynthesis.