Eclosion hormone (EH) is an insect neuropeptide that is released at the end of metamorphosis from the CNS and triggers the stereotyped motor program of adult emergence. Using three distinct experimental approaches, we have identified a discrete set of neurosecretory cells in the brain of the moth Manduca sexta that contains and releases EH. By isolating the neurosecretory somata and testing them with a sensitive behavioral bioassay, we identified a cluster of ipsilaterally projecting cells (Group Ia) that contain EH. Intracellular stimulation of individual cells within this group induced the release of bioactive EH into the hemolymph surrounding the neurohemal organs of the brain, whereas stimulation of cells in the other cerebral neurosecretory clusters did not. We also developed a polyclonal antiserum against purified EH that precipitated all bioactive material from samples containing the peptide. This antiserum selectively stained 5 of the Group Ia cells on either side of the brain, as well as their central and terminal processes. Preincubation of the serum with EH dramatically reduced its ability to bind the peptide subsequently. The combined application of these physiological and immunological techniques has led to the unequivocal identification of the EH neurons in the moth brain.