Biochemical, cytochemical, and physiological investigations have demonstrated the presence of the nitric oxide/cyclic GMP signaling system in the brain of the adult locust, Schistocerca gregaria. Here, we characterize nitric oxide (NO) releasing neurons and neurons that synthesize cyclic GMP (cGMP) in response to a NO stimulus in the brain of the embryonic grasshopper. Using NADPH-diaphorase histochemistry to detect NO synthesizing cells we describe the appearance of several individually identifiable neurons. At embryonic stage 50% four NADPH-diaphorase positive neurons can be detected in each brain hemisphere. In addition to the labeling of differentiating neurons, NADPH-diaphorase staining appears also in distinct proliferative cell clusters. At embryonic stage 70% the general organization of NADPH-diaphorase activity starts to resemble the adult brain. The immunocytochemical detection of NO-induced accumulation of cGMP starts at embryonic stage 45% resulting in the staining of large neuronal populations in all brain areas. During embryonic stages 50-70%, the number of cGMP-immunoreactive cells increases from 200 to several hundred in each brain hemisphere. Since all NADPH-diaphorase positive local interneurons of the adult antennal lobe express GABA-immunoreactivity, we also report on the earliest appearance of GABA-immunoreactivity in the embryonic antennal lobe. Thus, we present a first developmental investigation of nitrergic and GABAergic transmitter phenotypes in the brain of the embryonic grasshopper.