We examined the molecular basis of the circadian control of egg hatching behavior in the silkmoth Antheraea pernyi. Egg hatching is rhythmically gated, persists under constant darkness, and can be entrained by light by midembryogenesis. The time of appearance of photic entrainment by the silkmoth embryo coincides with the appearance of Period (PER) and Timeless (TIM) proteins in eight cells in embryonic brain. Although daily rhythms in PER and/or TIM immunoreactivity in embryonic brain were not detected, a robust circadian oscillation of PER immunoreactivity is present in the nuclei of midgut epithelium. per antisense oligodeoxynucleotide treatment of pharate larvae on the day before hatching consistently abolishes the circadian gate of egg hatching behavior. per antisense treatment also causes a dramatic decrease in PER immunoreactivity in newly hatched larvae. The results provide direct evidence that PER is a necessary element of a circadian system in the silkmoth.