HCN cation channel mRNA expression was determined in the rabbit heart and neonatal and adult rat ventricle using RNase protection assays. In the rabbit SA node, the dominant HCN transcript is HCN4, representing >81% of the total HCN message. HCN1 is also expressed, representing >18% of the total HCN mRNA. Rabbit Purkinje fibers contained almost equal amounts of HCN1 and HCN4 transcripts with low levels of HCN2, whereas rabbit ventricle contained predominantly HCN2. The SA node contained 25 times the total HCN message of Purkinje fibers and 140 times the total HCN message of ventricle. No reports of hyperpolarization-activated current (If) exist in rabbit Purkinje fibers, and we could not record If in rabbit ventricular myocytes. To investigate the possible role of isoform switching in determining the voltage dependence of If, we determined the prevalence of HCN isoforms in neonatal and adult rat ventricle. We had previously determined the threshold for activation of If to be approximately -70 mV in neonatal rat ventricle and -113 mV in adult rat ventricle. In both neonatal and adult rat ventricle, only HCN2 and HCN4 transcripts are present. The ratio of HCN2 to HCN4 is approximately 5:1 in the neonate and 13:1 in the adult. Taken together, these results suggest that different cardiac regions express different isoforms of the HCN family. The HCN1 and HCN4 isoforms are most closely associated with a depolarized threshold for If activation, whereas the HCN2 isoform is associated with a more negative activation curve.