Glutamate is considered to be the most likely transmitter candidate at excitatory synapses onto skeletal muscles of insects. We investigated the distribution of glutamate-like immunoreactivity (Glu-LI) in identified motor neurons of glutaraldehyde-fixed metathoracic ganglia of the locust in paraffin serial sections. The presumably glutamatergic fast and slow extensor tibiae motor neurons show Glu-LI, whereas other cells, including the GABAergic common inhibitory motor neurons and the cluster of octopaminergic dorsal unpaired median cells, show rather low levels of staining. Immunoreactivity of the fast extensor tibiae motor neuron is located in soma, neurites, axon, and the terminal arborizations. A double-labeling experiment on sections of the locust metathoracic ganglion showed that antisera against glutamate and GABA discriminate between the presumably glutamatergic and GABAergic motor neurons and that GABA-LI-positive neurons are low in Glu-LI. The results suggest that Glu-LI can be used as a marker for detecting potential glutamatergic neurons in insects under the present conditions. Application of the glutamate antiserum to sections of the honeybee brain revealed Glu-LI in motor neurons but also in certain interneurons. The most prominent populations of Glu-LI-positive cells were the monopolar cells and large ocellar interneurons, which are first-order interneurons of the visual and ocellar system. Several groups of descending interneurons also showed Glu-LI. The distributions of Glu-LI and GABA-LI are complementary in locust and bee ganglia. The high level of Glu-LI in certain interneuronal populations, as well as in identified glutamatergic motor neurons, suggests that insect central nervous systems may contain glutamatergic neuronal pathways.