Using an antiserum directed against glutamate, we have analyzed the distribution of glutamate-like immunoreactive neurons in the terminal abdominal ganglion of the crayfish Procambarus clarkii. Approximately 160 central neurons (157 +/- 8; mean +/- SEM, n = 8) showed positive glutamate-like immunoreactivity, which represents approximately 25% of the total number of neurons in the terminal ganglion. Using a combination of intracellular staining with the marker Lucifer yellow and immunocytochemical staining has shown that most excitatory motor neurons are glutamatergic and that glutamate acts as an excitatory transmitter at peripheral neuromuscular junctions. Seven of 10 identified spiking local interneurons and only 2 of 19 identified ascending interneurons, showed positive immunoreactivity. Our observation that inhibitory spiking interneurons were immunopositive, whereas excitatory ascending interneurons were immunonegative, indicates that glutamate is likely to act as an inhibitory neurotransmitter within the central nervous system. Local pressure injection of L-glutamate into the neuropil of the ganglion caused a hyperpolarization of the membrane potentials of many interneurons. gamma-Aminobutyric acid (GABA)ergic posterolateral nonspiking interneurons and the bilateral nonspiking interneuron LDS showed no glutamate-like immunoreactivity, whereas non-GABAergic anterolateral III nonspiking interneurons showed glutamate-like immunoreactivity. Thus, not only GABA but also glutamate are used in parallel as inhibitory neurotransmitters at central synapses. (C) 2004 Wiley-Liss, Inc.