Hyperpolarization-activated, cyclic nucleotide-gated cation (HCN) channels are powerful modulators of the membrane potential as well as the membrane resistance, and thus may play a major role in synaptic transmission and plasticity. Especially at the axon terminals of the medial perforant path (MPP) of the immature rat hippocampus, presynaptic expression of these channels has been demonstrated which declines upon maturation. The aim of this study was to elucidate the involvement of HCN channels in long-term depression (LTD) within the middle molecular layer comparing early postnatal (P9-15) and adult (P30-60) CD rats. Low-frequency stimulation (LFS; 900 pulses at 1 Hz) of the MPP-induced an immediate decrease (post-tetanic depression or PTD) in the amplitude of the field excitatory postsynaptic potentials (fEPSPs) resulting in stable LTD in slices from both animal groups, which sustained at least up to 60 min after LFS. Following bath application of specific HCN channel blocker ZD7288 (10 mM) prior to LFS, the same amount of PTD or LTD was obtained in slices from adult rats (P30-60) as compared to that obtained in control slices without drug perfusion (PTD with ZD7288: 81.2±5.1% vs. PTD control: 69.9±4.4%, P > 0.05; LTD with ZD7288: 84.9±4.5% vs. LTD control: 84.9±5.5%, P > 0.05, all data were normalized with interleaved slices without LFS). In marked contrast, in slices from early postnatal rats (P9–15), application of ZD7288 before LFS significantly enhanced both PTD and LTD levels as compared to slices in conditions without drug perfusion (PTD with ZD7288: 52.6±4.7% vs. PTD control: 68.9±5.8%, P < 0.05; LTD with ZD7288: 62.1±4.2% vs. LTD control: 80.4±4.2%; P < 0.01 after normalization). In another set of experiments, ZD7288 was applied after LFS. In these experiments, however, both PTD and LTD were no longer different from control slices without drug perfusion, but significantly less than the values obtained with ZD7288 applied before LFS (values for ZD7288 perfusion after LFS: PTD: 68.2±4.6%, P < 0.05 vs. control and vs. ZD7288 before LFS; LTD: 81.2±4.5%, P < 0.01 after normalization). In conclusion, the data presented here show that HCN channels at the medial perforant path-granule cell synapse impair LFS-induced LTD in early postnatal rats. Given the developmental decline of presynaptic HCN channels at this synapse as well as the lack of ZD7288 effect on LTD in slices of mature rats, we suggest that this HCN channel-mediated LTD impairment is due to modulation of presynaptic transmitter release.