Rhythmic activity of neurons and heart cells is endowed by pacemaker channels that are activated by hyperpolarization and directly regulated by cyclic nucleotides (termed HCN channels). These channels constitute a multigene family, and it is assumed that the properties of each member are adjusted to fit its particular function in the cell in which it resides. Here we report the molecular and functional characterization of a human subtype hHCN4. hHCN4 transcripts are expressed in heart, brain, and testis. Within the brain, the thalamus is the predominant area of hHCN4 expression. Heterologous expression of hHCN4 produces channels of unusually slow kinetics of activation and inactivation. The mean potential of half-maximal activation (V(1/2)) was -75.2 mV. cAMP shifted V(1/2) by 11 mV to more positive values. The hHCN4 gene was mapped to chromosome band 15q24-q25. The characteristic expression pattern and the sluggish gating suggest that hHCN4 controls the rhythmic activity in both thalamocortical neurons and pacemaker cells of the heart.