During metamorphosis, the insect nervous system undergoes considerable remodeling: new neurons are integrated while larval neurons are remodeled or eliminated. To understand further the mechanisms involved in transforming larval to adult tissue we have mapped the metamorphic changes in a particularly well established brain area, the antennal lobe of the sphinx moth Manduca sexta, using an antiserum recognizing RFamide-related neuropeptides. Five types of RFamide-immunoreactive (ir) neurons could be distinguished in the antennal lobe, based on morphology and developmental appearance. Four cell types (types II-V, each consisting of one or two cells) showed RFamide immunostaining in the larva that persisted into metamorphosis. By contrast, the most prominent group (type I), a mixed population of local and projection neurons consisting of about 60 neurons in the adult antennal lobe, acquired immunostaining in a two-step process during metamorphosis. In a first step, from 5 to 7 days after pupal ecdysis, the number of labeled neurons reached about 25. In a second step, starting about 4 days later, the number of RFamide-ir neurons increased within 6 days to about 60. This two-step process parallels the rise and fall of the developmental hormone 20-hydroxyecdysone (20E) in the hemolymph. Artificially shifting the 20E peak to an earlier developmental time point resulted in the precocious appearance of RFamide immunostaining and led to premature formation of glomeruli. Prolonging high 20E concentrations to stages when the hormone titer starts to decline had no effect on the second increase of immunostained cell numbers. These results support the idea that the rise in 20E, which occurs after pupal ecdysis, plays a role in the first phase of RFamide expression and in glomeruli formation in the developing antennal lobes. The role of 20E in the second phase of RFamide expression is less clear, but increased cell numbers showing RFamide-ir do not appear to be a consequence of the declining levels in 20E that occur during adult development.