Hyperpolarization-activated currents (Ih) are key players in shaping rhythmic neuronal activity. Although candidate genes for Ih channels have been cloned (HCN1-HCN4), the subunit composition of different native Ih channels is unknown. We used a combined patch-clamp and qualitative single-cell reverse transcription multiplex polymerase chain reaction (RT-mPCR) approach to analyse HCN1-4 coexpression profiles in four neuronal populations in mouse CNS. Coexpression of HCN2, HCN3 and HCN4 mRNA was detected in single neurons of all four neuronal cell types analysed. In contrast, HCN1 mRNA was detected in neocortical and hippocampal pyramidal neurons but not in dopaminergic midbrain and thalamocortical neurons. HCN1 expression was correlated with significantly faster activation kinetics on the level of individual neurons. Semiquantitative single-cell RT-mPCR analysis demonstrated that HCN1 mRNA expression is at least eightfold higher in cortical neurons than subcortical neurons. We show that single neurons possess complex coexpression patterns of Ih candidate genes. Alternative expression of HCN1 is likely to be an important molecular determinant to generate the different neuronal Ih channel species adapted to tune either subcortical or cortical network activity.