To investigate the relation between perforant path and the pattern of glutamate-like immunoreactivity in its target regions in the rat hippocampal formation, unilateral lesions of various size and location were placed to interrupt certain contingents of these afferent fibers. Postembedding immunohistochemistry at the level of light microscopy yielded the same pattern of immunoreactivity in the hippocampal formation contralateral to the lesion as in untreated animals. On the ipsilateral side, however, extensive transections of the perforant path led to a drastic loss of glutamate-immunoreactive terminal-like elements in the outer part of the dentate molecular layer. More restricted lesions induced a loss of punctate glutamate-like immunoreactivity in narrower bands within this zone. The width and the location of the affected bands appeared to depend on the extent of the transections and their topographical relation to the perforant path fiber system. These results and those obtained using a postembedding immunogold method at the level of electron microscopy strongly indicate that perforant path terminals in the dentate molecular layer of the rat contain high levels of glutamate and, thus, provide further support for an already well-documented role of this excitatory amino acid as neurotransmitter in this system.