Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed in the brain and heart and are essential for physiological functions in cardiac and nervous systems. We identified two Hcn4 mRNA variants with different transcription start sites and differential expression patterns in mouse brain and heart. Only one mRNA variant was detected in the brain, whereas both variants were found in the heart. Patch clamp recordings of these two variants in HEK293H cells revealed different electrophysiological properties in channel activation. Mutagenesis studies showed that three positively charged amino acids (Arg-9, Lys-10, and Lys-22) contribute to the functional difference. Our results demonstrate that HCN4 channels are expressed in different patterns in mouse brain and heart and that the N terminus is important for HCN4 channel activation.