ABSTRACT Allatotropin is a neuropeptide that was originally isolated from the sphinx moth Manduca sexta, in which it stimulates the synthesis of juvenile hormone in adults (Kataoka et al. 1989). An antiserum raised against this peptide was characterized, shown to be specific for allatotropin and used in a competitive enzyme-linked immunosorbent assay (ELISA) to demonstrate that allatotropin is present not only in the brain and retrocerebral complex but also in the ventral nerve cord of Manduca sexta (Veenstra and Hagedorn, 1993). In the present study, allatotropin-immunoreactive neurons were located using the allatotropin antiserum in a whole-mount immunofluorescence method adapted from Davis et al. (1989). Several cell types were found to be immunoreactive, including three pairs of intensely staining median neurons in abdominal ganglia 3–6 of the pharate adult. The location of these median pairs of cells in each ganglion is anterior, mid-dorsal and posterio-ventral (Fig. 1A). The cells project bilaterally via the ventral nerves of each ganglion to the next transverse nerve (Fig. 2), where their axons terminate in numerous superficial varicosities (Fig. 1D). It is well established that the transverse nerve is a neurohemal organ in Lepidoptera (Provansal, 1972; Taghert and Truman, 1982; Tublitz and Truman, 1985a–d). The location and projection pattern of these three pairs of median neurons indicate that they belong to a group of abdominal median neurosecretory cells identified previously (Taghert and Truman, 1982; Tublitz and Truman, 1985a). Using the nomenclature of Davis et al. (1993), these are the M1, M2 and M5 neurosecretory cells. The M1 and M2 cells mature during adult development, but the M5 cells are already differentiated in the larval stages (Taghert and Truman, 1982; Tublitz and Truman, 1985a). The M5 cells of larvae were not stained by the allatotropin antiserum and only became immunoreactive, along with the M1 and M2 cells, during adult metamorphosis. Two pairs of the abdominal median neurosecretory cells were not allatotropin-immunoreactive: (1) the M3 cells, which are ventral and mature during adult metamorphosis (Taghert and Truman, 1982); and (2) the M4 cells, which are already differentiated in the larval stages (Tublitz and Truman, 1985a).