Hyperpolarization-activated cation currents, termed I(h), are observed in a variety of neurons. Four members of a gene family encoding hyperpolarization-activated cyclic-nucleotide-gated cation channels (HCN1-4) have been cloned. The regional expression and cellular localization of the four HCN channel types in mouse brain was investigated using in situ hybridization. The expression of HCN1 was restricted to the olfactory bulb, cerebral cortex, hippocampus, superior colliculus and cerebellum. In contrast, HCN2 transcripts were found at high levels nearly ubiquitously in the brain, and the strongest signals were seen in the olfactory bulb, hippocampus, thalamus and brain stem. HCN3 was uniformly expressed at very low levels throughout the brain. Finally, HCN4 transcripts were prominently expressed selectively in the thalamus and olfactory bulb. Some neurons expressed two or more HCN channel transcripts including hippocampal pyramidal neurons (HCN1, HCN2 and low levels of HCN 4) and thalamic relay neurons (HCN2 and HCN4). Our results demonstrate that each HCN channel transcript has a unique distribution in the brain. Furthermore, they suggest that the heterogeneity of neuronal I(h) may be, at least in part, due to the differential expression of HCN channel genes.