In the normal granule cells of the dentate gyrus glutamate, GABA and glutamic acid decarboxylase (GAD67) coexist. After kindled seizures, this enzyme is transiently overexpressed and simultaneous glutamatergic and GABAergic transmission in the mossy fiber projection occurs. Since this dual transmission is also seen after acutely-induced seizures, we decided to study the relationship between the expression of GAD67 and the induction of simultaneous glutamatergic and GABAergic transmission by kindled or acutely induced seizures. We also explored whether kindling of the dentate gyrus in vitro, that does not induce epileptiform activity, could induce the expression of GAD67. We confirm that kindling epilepsy induces the expression of GAD67 in the dentate gyrus. Despite the emergence of GABAergic transmission in the mossy fiber projection after a single seizure, GAD67 expression in the dentate gyrus appeared similar to controls, however, in the mossy fibers an enhanced immunostaining was evident. Interestingly, kindling the dentate gyrus in vitro induces a marked GAD67 staining in the granule cells. Our results show that after the activity-dependent emergence of simultaneous glutamatergic and GABAergic transmission from the mossy fibers, GAD67 is expressed in the mossy fibers and, upon long-lasting enduring stimulation periods, also in the dentate gyrus. Thus, this phenomenon does not depend on the presence of epileptic activity, but rather, on increased excitatory input onto the dentate gyrus. This can represent a protective mechanism that can sustain GABA synthesis in an activity-dependent manner.