Electrophysiologically identified and intracellularly biocytin-labeled mossy cells in the dentate hilus of the rat were studied using electron microscopy and postembedding immunogold techniques. Ultrathin sections containing a labeled mossy cell or its axon collaterals were reacted with antisera against the excitatory neurotransmitter glutamate and against the inhibitory neurotransmitter gamma-aminobutyric acid (GABA). From single- and double-immunolabeled preparations, we found that 1) mossy cell axon terminals made asymmetric contacts onto postsynaptic targets in the hilus and stratum moleculare of the dentate gyrus and showed immunoreactivity primarily for glutamate, but never for GABA; 2) in the hilus, glutamate-positive mossy cell axon terminals targeted GABA-positive dendritic shafts of hilar interneurons and GABA-negative dendritic spines; and 3) in the inner molecular layer, the mossy cell axon formed asymmetric synapses with dendritic spines associated with GABA-negative (presumably granule cell) dendrites. The results of this study support the view that excitatory (glutamatergic) mossy cell terminals contact GABAergic interneurons and non-GABAergic neurons in the hilar region and GABA-negative granule cells in the stratum moleculare. This pattern of connectivity is consistent with the hypothesis that mossy cells provide excitatory feedback to granule cells in a dentate gyrus associational network and also activate local hilar inhibitory elements.