The hyperpolarization-activated cation current (I(h)), mediated by HCN channels, contributes to intrinsic neuronal properties, synaptic integration and network rhythmicity. Recent studies have implicated HCN channels in neuropathological conditions including epilepsy. While native HCN channels have been studied at the macroscopic level, the biophysical characteristics of individual neuronal HCN channels have not been described. We characterize, for the first time, single HCN currents of excised inside-out patches from somata of acutely dissociated rat hippocampal CA1 pyramidal cells. Hyperpolarization steps elicited non-inactivating channel openings with an apparent conductance of 9.7 pS, consistent with recent reports of native and recombinant HCN channels. The voltage-dependent P(o) had a V(1/2) of -81 +/- 1.8 mV and slope -13.3 +/- 1.9 mV. Blockers of macroscopic I(h), ZD7288 (50 microM) and CsCl (1 mM), reduced the channel conductance to 8 pS and 8.4 pS, respectively. ZD7288 was slightly more effective in reducing the P(o) at depolarized potentials, whereas CsCl was more efficacious at hyperpolarized potentials. The unitary neuronal HCN channels had voltage-dependent latencies to first channel opening and two open states. As expected, ZD7288 and CsCl increased latencies and decreased the properties of both open states. The major endogenous positive modulator of macroscopic I(h) is cAMP. Application of 8Br-cAMP (10 microM) did not affect conductance (9.4 pS), but did increase P(o) and short and long open times. Thus, sensitivity to I(h) modulators supports the single h-channel identity of these unitary currents. Detailed biophysical analysis of unitary I(h) conductances is likely to help distinguish between homomeric and heteromeric expression of these channels - findings that may be relevant toward the pathophysiology of diseases such as epilepsy.