Mossy fiber sprouting has been proposed to lead to new excitatory connections between dentate granule cells, which in turn cause electrographic seizures. We tested this hypothesis in hippocampal slices from rats made epileptic-by kainate injections. The Timm's histological method revealed intense staining of the inner molecular layer in slices from all kainate-treated rats. In bicuculline (10 microM) and 6 mM [K +]o, antidromic stimulation of the granule cells evoked bursts of population spikes superimposed on long-lasting negative shifts in all slices tested from all kainate-treated rats. Long-duration (2-47 sec), seizure-like bursts with tonic and clonic components occurred spontaneously (53%) or in response to antidromic stimulation (81%). Under identical conditions, prolonged bursts were never seen in slices from controls or from kainate-injected rats 2-4 d after treatment. Glutamate microdrops applied in the granule cell layer evoked abrupt increases in the frequency of excitatory postsynaptic potentials (EPSPs) in two thirds of the cells tested. Glutamate microstimulation was effective at several sites in the granule cell layer but ineffective in the hilus. The proportion of granule cells responding to local application of glutamate by an increase in EPSPs was higher in slices with long bursts (80% with bursts of > 3 sec) than in slices with shorter bursts (33% with bursts of < 3 sec). Glutamate microstimulation did not affect EPSPs in granule cells from control preparations. These results support the hypothesis that kainate-induced mossy fiber sprouting forms new excitatory connections between granule cells and can lead to increased seizure susceptibility in the dentate gyrus.