Using organ culture, high-resolution liquid chromatography, and microchemical techniques, we demonstrated the efficient incorporation in vitro of several radiolabeled precursors into the two juvenile hormones of Manduca sexta. JH II, a homosesquiterpene hormone, reported from M. sexta as well as several other insects, incorporates radiolabel from acetate, mevalonate, and propionate. JH III, a sesquiterpene hormone recently reported as a natural product of M. sexta, incorporates label from acetate and mevalonate, but not from propionate. Based on the position of the labeled atoms in the precursors and upon the position of incorporation obtained from label-distribution data, a scheme for juvenile hormone biosynthesis is advanced.