We have mapped protein expression of the FMRFamide neuropeptide gene in Drosophila with polyclonal antisera against three small peptides whose sequences were derived from the Drosophila proFMRFamide precursor. One antiserum was affinity-purified and extensively characterized. The enriched antibodies labeled 15-21 bilaterally symmetric pairs of neurons in a pattern that corresponded very closely to the pattern of in situ hybridization that was determined previously (Schneider et al. [1991] J. Comp. Neurol. 304:608-622; O'Brien et al. [1991] J. Comp. Neurol. 304:623-638). The other antisera produced complementary results. These findings suggest that the antisera specifically label cells that express the FMRFamide gene. In larvae we consistently observed strong staining in identified interneurons and neuroendocrine cells, and moderate to weak staining in neurons of unknown function. The adult pattern of expression included both larval neurons whose immunoreactivity persisted through metamorphosis and adult-specific neurons. During metamorphosis, we observed transient staining in a small number of neurons and in specific neuropil regions that included the central body, the protocerebral bridge, and the optic ganglia. Based on these morphological features, we suggest that the FMRFamide-like neuropeptides in Drosophila play a number of functional roles, perhaps affecting both physiological and developmental phenomena. Such roles include general modulation throughout all post-embryonic stages, via the blood, and also more stage- and region-specific modulation within the CNS.