Voltage-clamp studies of freshly isolated smooth muscle cells from rabbit portal vein revealed the existence of a time-dependent cation current evoked by membrane hyperpolarization (termed I_h). Both the rate of activation and the amplitude of I_h were enhanced by membrane hyperpolarization. Half-maximal activation of I_h was about 105 mV with conventional whole cell and 80 mV when the perforated patch technique was used. In current clamp, injection of hyperpolarizing current produced a marked depolarizing "sag" followed by rebound depolarization. Activation of I_h was augmented by an increase in the extracellular K⁺ concentration and was blocked rapidly by externally applied Cs⁺ (1-5 mM). The bradycardic agent ZD-7288 (10 µM), a selective inhibitor of I_h, produced a characteristically slow inhibition of the portal vein I_h. The depolarizing sag recorded in current clamp was also abolished by application of 5 mM Cs⁺. Cs⁺ significantly decreased the frequency of spontaneous contractions in both whole rat portal vein and rabbit portal vein segments. Multiplex RT-PCR of rabbit portal vein myocytes using primers derived from existing genes for hyperpolarization-activated cation channels (HCN1-4) revealed the existence of cDNA clones corresponding to HCN2, 3, and 4. The present study shows that portal vein myocytes contain genes shown to encode for hyperpolarization-activated channels and exhibit an endogenous current with characteristics similar to I_h in other cell types. This conductance appears to determine, in part, the rhythmicity of this vessel.