The excitatory, glutamatergic granule cells of the hippocampal dentate gyrus are presumed to play central roles in normal learning and memory, and in the genesis of spontaneous seizure discharges that originate within the temporal lobe. In localizing the two GABA-producing forms of glutamate decarboxylase (GAD65 and GAD67) in the normal hippocampus as a prelude to experimental epilepsy studies, we unexpectedly discovered that, in addition to its presence in hippocampal nonprincipal cells, GAD67-like immunoreactivity (LI) was present in the excitatory axons (the mossy fibers) of normal dentate granule cells of rats, mice, and the monkey Macaca nemestrina. Using improved immunocytochemical methods, we were also able to detect GABA-LI in normal granule cell somata and processes. Conversely, GAD65-LI was undetectable in normal granule cells. Perforant pathway stimulation for 24 hours, which evoked population spikes and epileptiform discharges in both dentate granule cells and hippocampal pyramidal neurons, induced GAD65-, GAD67-, and GABA-LI only in granule cells. Despite prolonged excitation, normally GAD- and GABA-negative dentate hilar neurons and hippocampal pyramidal cells remained immunonegative. Induced granule cell GAD65-, GAD67-, and GABA-LI remained elevated above control immunoreactivity for at least 4 days after the end of stimulation. Pre-embedding immunocytochemical electron microscopy confirmed that GAD67- and GABA-LI were induced selectively within granule cells; granule cell layer glia and endothelial cells were GAD- and GABA-immunonegative. In situ hybridization after stimulation revealed a similarly selective induction of GAD65 and GAD67 mRNA in dentate granule cells. Neurochemical analysis of the microdissected dentate gyrus and area CA1 determined whether changes in GAD- and GABA-LI reflect changes in the concentrations of chemically identified GAD and GABA. Stimulation for 24 hours increased GAD67 and GABA concentrations sixfold in the dentate gyrus, and decreased the concentrations of the GABA precursors glutamate and glutamine. No significant change in GAD65 concentration was detected in the microdissected dentate gyrus despite the induction of GAD65-LI. The concentrations of GAD65, GAD67, GABA, glutamate and glutamine in area CA1 were not significantly different from control concentrations. These results indicate that dentate granule cells normally contain two "fast-acting" amino acid neurotransmitters, one excitatory and one inhibitory, and may therefore produce both excitatory and inhibitory effects. Although the physiological role of granule cell GABA is unknown, the discovery of both basal and activity-dependent GAD and GABA expression in glutamatergic dentate granule cells may have fundamental implications for physiological plasticity presumed to underlie normal learning and memory. Furthermore, the induction of granule cell GAD and GABA by afferent excitation may constitute a mechanism by which epileptic seizures trigger compensatory interictal network inhibition or GABA-mediated neurotrophic effects.