At the end of each instar, insects shed their old cuticle by performing the stereotyped ecdysis behavior. In the month, Manduca sexta, larval ecdysis is accompanied by increases in intracellular cyclic 3', 5'-guanosine monophosphate (cGMP) in a small network of 50 peptidergic neurons within the ventral central nervous system (CNS). Studies on a variety of insects show that this cGMP response has been associated with ecdysis throughout most of insect evolution. In the mealbeetle (Tenebrio, Coleoptera) and the mosquito (Aedes, Diptera), all 50 neurons showed increases in cGMP immunoreactivity (-IR) at ecdysis, and all were immunopositive for crustacean cardioactive peptide (CCAP). Other insects varied with respect to their cGMP response at ecdysis and their CCAP-IR. In more primitive insects, such as the silverfish (Ctenolepisma, Zygentoma) and the grasshopper (Locusta, Orthoptera), an abdominal subset of these neurons did not show detectable cGMP-IR at ecdysis, although the neurons were CCAP-IR. Conversely, whereas CCAP-IR was severely reduced in the thoracic and subesophageal neurons of Lepidoptera larvae and may be absent in a subset of the corresponding abdominal neurons in crickets (Gryllus, Orthoptera), the ecdysial cGMP response occurred in all 50 neurons. The most extreme case was found in cyclorrhaphous flies, in which most of the 50 neurons were CCAP-IR, although none showed increases in cGMP at ecdysis. This situation in higher Diptera is discussed in terms of their highly modified ecdysis behaviors.