Pupal diapause in the tobacco hornworm Manduca sexta is a photoperiodically induced event expressed by the absence of release of the cerebral peptide prothoracicotropic hormone. A short-day photoperiod (light-dark, 12:12) during larval development programs the brain to forego the release of prothoracicotropic hormone in the pupal stage. Prothoracicotropic hormone release can occur in the pupae and diapause can be averted if developing larvae experience an intervening long-day photoperiod (light-dark, 16:8) for 3 days early in the last larval instar. Implanted brains from larvae that received such long-day stimulation can reverse the diapause commitment of short-day-reared diapause-destined larvae, thus overriding the diapause program of the recipient. A system was established that demonstrated that this photoperiodic reprogramming of the insect's brain occurs in vitro. Brain-retrocerebral complexes from day 1 instar larvae were cultured in vitro for 3 days under long-day conditions. The effect of this non-diapause photoperiod on the diapause program of these brain-retrocerebral complexes was assessed by assaying the effect of the brains from these cultures on the incidence of non-diapause development in diapause-destined larval hosts. Brains that received a long-day photoperiod reversed the pupal diapause program of the recipient larvae, whereas brains that received a short-day photoperiod were ineffective. Therefore, the larval brain-retrocerebral complex of this insect is capable of photoperiodic light reception in vitro, in that it can sense the number of light-dark cycles to which it is exposed, store this environmental input, and express the information 9 days later at another stage of development. These findings demonstrate that the photoperiodic clock of an animal can be reprogrammed by a light-dark cycle in vitro.