The cation conductance in primary cultures of rat renal inner medullary collecting duct was studied using perforated-patch whole-cell clamp and conventional whole-cell clamp techniques. Hyperpolarizations beyond -60 mV induced a time-dependent inward nonselective cationic current (I_vti) that resembles the well known hyperpolarization-activated, cyclic nucleotide-gated I_h and I_f currents. I_vti showed a half maximal activation around -102 mV with a slope factor of 25 mV. It had a higher conductance (but, at its reversal potential, not a higher permeability) for K⁺ than for Na⁺, was modulated by cAMP, blocked by external Cd²⁺ (but not Cs+ or ZD7288), and potentiated by a high extracellular K⁺ concentration. We explored the expression of the I_h channel genes (HCN1 to 4) by RT-PCR. The presence of transcripts corresponding to the HCN1, 2 and 4 genes was observed both in the cultured cells and kidney inner medulla. Western blot analysis with HCN2 antibody showed labeling of ~90 and ~120 kDa proteins in samples from inner medulla and cultured cells. Immunocytochemical analysis of cell cultures and inner medulla showed the presence of HCN immunoreactivity partially colocalized with the Na⁺/K⁺ ATPase at the basolateral membrane of collecting duct cells. This is the first evidence of an I_h-like cationic current and HCN immunoreactivity in either kidney or any other nonexcitable mammalian cells.