1. The fates of the 22-pro-R and 22-pro-S hydrogen atoms of cholesterol during the biosynthesis of ecdysteroids in the ovaries of Schistocerca gregaria were investigated. 2. Two stereospecifically labelled cholesterol species, obtained by incubating 3R,2R- and 3R,2S-[2-14C, 2-3H]mevalonic acid with rat liver preparations, were administered, in turn, to maturing adult female locusts and the radiolabelled ecdysteroid conjugates isolated from the eggs. Enzymic hydrolysis of the conjugates yielded free ecdysteroids, from which ecdysone was purified. 3. Derivative formation and oxidation at C-22 of both ecdysone samples indicated that the 22-pro-R and 22-pro-S hydrogen atoms of cholesterol were stereospecifically eliminated and retained respectively during ecdysteroid formation. This indicates that C-22 hydroxylation in ecdysone biosynthesis is direct and occurs with retention of configuration.