Recently, both nitric oxide synthase (NOS), and nitric oxide (NO)-sensitive guanylyl cyclase were cloned in Manduca sexta and implicated in several cellular, developmental, and behavioral processes (Nighorn et al. [1998] J Neurosci 18:7244 -7255). However, NO is a highly diffusive gas, and little is known about the range and specificity of its actions on neurons. To begin examining the role of NO as a neurotransmitter in the central nervous system (CNS) of larval Manduca, we have mapped potential NO-producing neurons using fixation-resistant NADPH-diaphorase staining and antisera that recognize a NOS-specific epitope. In addition, to detect NO-responsive neurons, we treated the CNS with NO donors and used antibodies that recognize elevated levels of cyclic 3Π,5Π-guanosine monophosphate (cGMP). Many potential NO-producing neurons were mapped, including the ventral unpaired median cells and three pairs of lateral cells in each abdominal ganglion. Additional neurons in the dorsal midline of ganglia A5-7 (PM2) appear to express NOS in a segment-specific manner. At the larval-to-pupal transition, this staining pattern changes; the PM2 neurons stain weakly or are undetectable and there is novel expression of NOS in cell 27. In response to NO donors, a small number of neurons produce detectable cGMP accumulation in a segment-specific pattern. These include a pair of posteriodorsally positioned interneurons (IN505) in all the abdominal ganglia, PM2 neurons in A5, and PM1 and PM2 neurons in A7. Hence, PM2 neurons in A5 and A7 are potentially capable of producing and responding to NO. These identified NO-producing and responding neurons provide a tractable model system for studying the dynamics and specificity of NO signaling in the CNS.