The action potential in gallbladder smooth muscle (GBSM) is caused by Ca²⁺ entry through voltage-dependent Ca²⁺ channels (VDCC), which contributes to the GBSM contractions. Action potential generation in GBSM is critically dependent on the resting membrane potential (about -50 mV), which is approximately 35 mV more positive of the K⁺ equilibrium potential. We hypothesized that a tonic, depolarizing conductance is present in GBSM and contributes to the regulation of the resting membrane potential and action potential frequency. GBSM cells were isolated from guinea pig gallbladders and the whole cell patch-camp technique was used to record membrane currents. After eliminating the contribution of VDCC and K⁺ channels, we identified a novel spontaneously active cation conductance (I_cat) in GBSM. This I_cat was mediated predominantly by influx of Na⁺ ions. Na⁺ substitution with N-methyl-D-glucamine (NMDG), a large relatively impermeant cation, caused a negative shift in the reversal potential of the ramp current and reduced the amplitude of the inward current at -50 mV by 65%. Membrane potential recordings with intracellular microelectrodes or in current clamp mode of the patch clamp technique indicated that the inhibition of I_cat conductance by NMDG is associated with membrane hyperpolarization and inhibition of action potentials. Extracellular Ca²⁺, Mg²⁺ and Gd³⁺ attenuated the I_cat in GBSM. Muscarinic stimulation did not activate the I_cat. Our results indicate that in GBSM, a Na⁺-permeable channel contributes to the maintenance of the resting membrane potential and action potential generation, and therefore plays a critical role in the regulation of GBSM excitability and contractility.