Three insect peptide hormones, eclosion hormone (EH), ecdysis-triggering hormone (ETH) and crustacean cardioactive peptide (CCAP), have been implicated in controlling ecdysis behavior in insects. This study examines the interactions between these three peptides in the regulation of the ecdysis sequence. Using intracellular recordings, we found that ETH is a potent activator of the EH neurons, causing spontaneous action potential firing, broadening of the action potential and an increase in spike peak amplitude. In turn, electrical stimulation of the EH neurons or bath application of EH to desheathed ganglia resulted in the elevation of cyclic GMP (cGMP) levels within the Cell 27/704 group (which contain CCAP). This cGMP production increases the excitability of these neurons, thereby facilitating CCAP release and the generation of the ecdysis motor program. Extracellular recordings from isolated nervous systems show that EH has no effect on nervous systems with an intact sheath. In desheathed preparations, in contrast, EH causes only the ecdysis motor output. The latency from EH application to ecdysis was longer than that after CCAP application, but shorter than that when ETH is applied to a whole central nervous system. These data, along with previously published results, support a model in which ETH causes pre-ecdysis behavior and at higher concentrations stimulates the EH neurones. EH release then facilitates the onset of ecdysis by enhancing the excitability of the CCAP neurons.