Changes in the gating properties of Na⁺ channels were studied in ES-derived neuronal stem (NS) cells during in vitro neuronal differentiation with the use of the whole-cell and cell-attached variants of patch-clamp technique. NS cells represent a novel stem cell population remaining stable and highly neurogenic over multiple passages. Voltage-clamp recordings during neuronal differentiation of NS cells indicated significant changes in the key properties of Na⁺ currents. A voltage-gated and tetrodotoxine-sensitive Na⁺ current, absent under self-renewal conditions, was first recorded following application of differentiative agents. Current density increased with time of exposure to differentiating conditions. Whole-cell and single channel analysis revealed that the observed increase in current density was due at least in part to changes in steady-state activation and inactivation properties. Namely, half activation potential shifted from -34 mV to -45 mV, while half-inactivation potential shifted from -94 mV to -78 mV. Furthermore, a contribution to the increase in Na⁺ current density, measured under whole-cell conditions, could also be given by an enhancement in channel expression, as suggested by an augmentation in the number of single channels per patch area, with increasing neuronal differentiation. Interestingly, those changes in Na⁺ channel activity well correlate with the capability of NS cells to generate action potentials during in vitro neuronal differentiation.