Hyperpolarization-activated Cation Non-selective (HCN) channels are voltage-gated ion channels that open at potentials more negative to -50 mV. In the hippocampus and cortex, these are known to be localized to pyramidal cell dendrites. However, emerging evidence suggests that these channels may also be present pre-synaptically in these areas. In support, our recent work (Huang et al., J. Neurosci., 2009) shows that excitatory synaptic transmission is elevated in the entorhinal cortex of HCN1 null mice, indicating that HCN1 channels may be present pre-synaptically. In this study, we have further investigated this possibility. Entorhinal-hippocampal cortical slices were prepared from adult (6-8 week old) HCN1 null mice and their wildtype littermates. Whole-cell voltage-clamp recordings were made from wildtype and HCN1-/- EC neurons. To reduce the contribution of post-synaptic HCN channels, the selective blocker, ZD7288 (15 μM) was included in the patch pipette. In the presence of GABA receptor blockers and tetrodotoxin (TTX), spontaneous miniature excitatory post-synaptic potential (mEPSC) frequency recorded from HCN1-/- EC layer III pyramid soma (5.76 ± 0.4, n=92) and dendrites (3.84 ± 0.6, n=5) was substantially greater than that obtained from wildtype soma (2.10 ± 0.1, n=115, p < 0.01) and dendrites (2.86 ± 0.3, n= 8). mEPSC amplitudes and kinetics, however, did not differ. Moreover, bath-application of ZD7288 (15 µM) doubled somatic and dendritic mEPSC frequency in wild-types whilst have no effect on HCN1-/- mEPSC frequency. In the absence of TTX, ZD7288 also reduced the paired pulse ratios of EPSCs evoked by stimulating the distal dendrites of EC layer III pyramids. Interestingly, mEPSC frequency, amplitudes, rise time constants and decay time constants obtained from wildtype and HCN1-/- EC layer II stellate and layer V pyramid soma was similar. Further, ZD7288 (15 μM) had little effect on wildtype mEPSCs in EC layer II stellate and layer V pyramid soma. In addition, miniature inhibitory post-synaptic currents (mIPSCs) recorded in the presence of glutamate receptor blockers and TTX from wildtype and HCN1-/- EC layer III pyramids were similar in frequency, amplitude and kinetics. In agreement with the electrophysiological results, electron microscopy revealed that HCN1 subunits were localized on excitatory presynaptic terminals predominantly located in EC layer III. Thus, these findings indicate that HCN1 channels may be present presynaptically at select glutamatergic synaptic terminals, where they exclusively modulate excitatory synaptic transmission.