The conversion of ecdysone (E) to 20-hydroxyecdysone (20E), the active arthropod molting hormone, via the cytochrome P450-dependent enzyme, ecdysone 20-monooxygenase (E20M), initiates the metabolic and physiological events that occur during molting and metamorphosis throughout insect postembryonic development. The present study used an in vitro radioassay to examine E20M enzyme activity throughout the 120-hour period of embryogenesis in the tobacco hornworm, Manduca sexta. E20M enzyme activity increases significantly at two points during embryogenesis, at 12 hours and 72 hours into embryogenesis. At 72 hours, E20M activity achieves its maximal level of activity observed during embryogenesis, subsequently decreasing to basal levels for the rest of the period. Interestingly, the 72-hour peak in enzyme activity corresponds to the time at which the accumulation of the 20-hydroxylated ecdysteroid observed in embryogenesis is at its peak, and this is the period between the secretion of the first and second embryonic cuticle.