Septohippocampal cholinergic neurons innervate the hippocampus and provide it with almost its entire acetylcholine. Axon collaterals of these neurons also release acetylcholine within the septum and thereby maintain the firing activity of septohippocampal GABAergic neurons. A loss of septohippocampal cholinergic neurons occurs in various neurodegenerative disorders associated with cognitive dysfunctions. Group I metabotropic glutamate receptors have been implicated in septohippocampal-dependent learning and memory tasks. In the present study, we examined the physiological and pharmacological effects of a potent and selective group I metabotropic glutamate receptor (mGluR) agonist DHPG, on rat septohippocampal cholinergic neurons that were identified in brain slices using a selective fluorescent marker. In whole-cell recordings, DHPG produced a reversible, reproducible and a direct post-synaptic and concentration-dependent excitation in 100% of septohippocampal cholinergic neurons tested with an EC₅₀ of 2.1 µM. Pharmacologically, the effects of DHPG were partially/completely reduced by the mGluR1 antagonists, CPCCOEt and LY367385. Addition of the mGluR5 antagonist, MPEP, reduced the remaining response to DHPG, suggesting involvement of both receptor subtypes in a subpopulation of septohippocampal cholinergic neurons. In double immunolabeling studies, 74% of septohippocampal cholinergic neurons co-localized mGluR1-immunoreactivity and 35% co-localized mGluR5-immunoreactivity. Double immunolabeling studies at the light and electron-microscopic levels showed that vesicular glutamate transporter 2 terminals make asymmetric synaptic contacts with septohippocampal cholinergic neurons. These findings may be of significance in treatment of cognitive deficits associated with neurodegenerative disorders as a Group I mGluR-mediated activation of septohippocampal cholinergic neurons would enhance the release of acetylcholine both in the hippocampus and in the septum.