The hippocampus contains one very strong recurrent excitatory network formed by associational connections between CA3 pyramidal cells and another that depends largely on a disynaptic excitatory pathway between dentate granule cells. The recurrent excitatory network in CA3 has long been considered a possible location of autoassociative memory storage, whereas changes in the level and arrangement of recurrent excitation between granule cells are strongly implicated in epileptogenesis. Hilar mossy cells are likely to receive collateral input from CA3 pyramidal cells and they are key intermediaries (by mossy fiber inputs) in the recurrent excitatory network between granule cells. The current study uses minimal stimulation techniques in an in vitro preparation of the rat dentate gyrus to examine presynaptic modulation of both mossy fiber and non-mossy fiber inputs to hilar mossy cells. We report that both mossy fiber and non-mossy fiber inputs to hilar mossy cells express presynaptic gamma-aminobutyric acid type B (GABA(B)) receptors that are subject to tonic inhibition by ambient GABA. We further find that only non-mossy fiber inputs express presynaptic muscarinic acetylcholine receptors, but that bath application of cholinergic agonists produces action potential-dependent increases in ambient GABA that can indirectly inhibit mossy fiber inputs. Finally, we demonstrate that mossy cells express high-affinity postsynaptic GABA(A) receptors that are also capable of detecting changes in ambient GABA produced by cholinergic agonists. Our results are among the first to directly characterize these important collateral inputs to hilar mossy cells and may help facilitate informed comparison between primary and collateral projections in two major excitatory pathways.