n the moth Manduca sexta, the declining ecdysteroid titer on the final day of the molt from the fourth to the fifth larval instar acts on the ventromedial neurosecretory cells (VM cells) to stimulate the release of eclosion hormone (EH). EH then triggers the motor programs involved in ecdysis behavior. Intracellular recordings that were made from the VM cells throughout the intermolt and molting stages showed no spontaneous action potentials until 0.9 hr before ecdysis (during the expected time of EH release), when 50% of the VM cells fired tonically at rest. This change was associated with a marked reduction in VM cell threshold without alteration of input resistance, resting potential, or synaptic drive. The increase in VM cell excitability was dependent on the declining ecdysteroid titer, because the injection of 20- hydroxyecdysone (20-HE) 11.5 hr before ecdysis significantly delayed the expected decrease in VM cell threshold. Since the same steroid treatment given 4.6 hr before ecydysis did not delay the subsequent increase in VM cell excitability, the inhibitory actions of 20-HE appear not to be mediated by a rapid membrane mechanism. The possible involvement of genomic events in the steroid-dependent increase in VM cell excitability was examined using the RNA synthesis inhibitor actinomycin D (AcD). When injected 6.3 hr before ecdysis, AcD blocked EH release without altering the response of the nervous system to exogenous peptide. Such AcD treatments also prevented the increase in VM cell excitability. These results suggest that the declining ecdysteroid titer increases the excitability of the VM cells via a transcription-dependent process.