Abstract Leg-amputation experiments performed on populations of carefully staged last-instar Rhodnius prolixus indicate that the regeneration critical period in this species occurs 6–7 days following feeding. Decapitation experiments indicate that the regeneration critical period is also the time of the head critical period for moulting. The head critical period is associated with a rapid increase in ecdysteroid titre in response to a release of prothoracicotropic hormone by the brain. Leg amputations performed prior to the head critical period result in a temporary decrease in ecdysteroid titre lasting 24–36 h, while the same operation performed after the head critical period has no effect on ecdysteroid titre. When the period of temporary depression in titre encompasses the head critical period, the rapid increase in ecdysteroid titre associated with this time is delayed. Thus, the ability to regenerate a leg after initiation of the moult is dependent upon when the injury occurs in relation to the release of prothoracicotropic hormone at the head critical period. The mechanism responsible for the decline in ecdysteroid titre following leg amputation is unknown but does not appear to involve prothoracicotropic hormone or the prothoracic glands. Injection of tetrodotoxin at the time of a leg amputation prolongs the period for which the titre is depressed. In these animals the titre rises in conjunction with resumption of electrical activity in the corpus cardiacum, indicating that prothoracicotropic hormone release may be necessary for recovery of the titre following leg amputation. Results are discussed in relation to existing evidence concerning possible interactions between regeneration, prothoracicotropic hormone release and moulting.