Cholinergic innervation of the hippocampus has been implicated in memory formation and retrieval. Here we study cholinergic modulation of excitatory transmission in the CA3 area of the rat hippocampus. We used a combination of optical measurements of presynaptic calcium and electrophysiological measurements of synaptic currents to study associational-commissural (A/C) and mossy fiber (MF) synapses in brain slices. Direct synaptic modulation mediated by ACh receptors is only evident at the A/C synapse, where synaptic inhibition primarily reflects presynaptic calcium channel inhibition mediated by muscarinic receptors. MF synapses can, however, be indirectly modulated by muscarinic receptor activation. Muscarine elevates the firing rate of inhibitory cells, which increases GABA release and inhibits MF synapses by activating presynaptic GABA(B) receptors. Muscarine also depolarizes dentate granule cells and elevates their rate of firing. This leads to synaptic enhancement when combined with the use-dependent facilitation of MF synapses. In addition we were unable to evoke an increase in presynaptic calcium levels in MF boutons with local application of nicotinic receptor agonists. This finding does not support a leading hypothesis for MF modulation in which activation of presynaptic nicotinic receptors enhances transmission directly by elevating presynaptic calcium levels. However, indirect synaptic modulation could arise from nicotinic excitation of inhibitory neurons. Thus, to understand cholinergic modulation within the CA3 region, it is necessary to take into account secondary actions on synapses arising from other chemical messengers released by other cell types and to consider effects on firing patterns of presynaptic cells, which in turn influence release via use-dependent synaptic plasticity.