Hyperpolarization-activated cyclic-nucleotide-gated (HCN) channels conduct a monovalent cationic current, I(h), which contributes to the electrophysiological properties of neurons and regulates thalamic oscillations in circuits containing the glutamatergic ventrobasal complex (VB) and GABAergic reticular thalamic nucleus (RTN). Four distinct HCN channel isoforms (HCN1-4) have been identified, and mRNAs and proteins for HCN channels have been detected in the RTN and VB, with HCN2 and HCN4 being the predominant isoforms. RTN and VB neurons have distinct electrophysiological properties, and those differences may reflect variable compartmental distribution of HCN channels. Whole cell patch clamp recordings from thalamic neurons in brain slices obtained from C57/Bl6 mice demonstrate that I(h) is much smaller in RTN than in VB neurons although the time constants for I(h) current activation are very similar. To study the compartmental distribution of the underlying channels, we performed qualitative and quantitative examination of HCN2 and HCN4 expression using fluorescent immunohistochemistry and confocal microscopy. HCN2-immunoreactivity (IR) on the somata of RTN neurons was approximately 10-fold less than that seen in VB neurons while HCN4-IR was detected on the somata of RTN and VB neurons to an equal degree. HCN2-IR in RTN and VB did not overlap with synaptophysin-IR, but strongly colocalized with cortactin-IR, indicating that HCN2 was not present in axon terminals but was present in dendritic spines. Although HCN2-IR in spines was more pronounced in VB than in RTN, the ratio of spinous to somatic expression in RTN was dramatically higher than that in VB, strongly suggesting that HCN2-IR in RTN is principally located in sites distal to the soma. In contrast, HCN4-IR did not colocalize with either synaptophysin or cortactin. The colocalization of HCN2-IR with HCN4-IR was greater in VB than in RTN. The results suggest that the distinct compartmental distribution of HCN2 channels in RTN and VB neurons contributes to the profound differences in the I(h)-dependent properties of these cells.