The causes of attenuation of Na⁺ currents by diacylglycerol (DAG)-induced protein kinase C (PKC) activation in mouse neuroblastoma N1E-115 cells were investigated using the cell-attached patch, and the perforated-patch (nystatin based) whole-cell voltage-clamp techniques. Activation of PKC by DAG attenuated Na⁺ currents. Attenuation occurred in the absence of significant changes in the time-course of Na⁺ currents. However, the steady-state inactivation curve of these currents shifted to more negative voltages by approximately 20 mV. Here we demonstrate that the time-course of inactivation is accelerated by treatment with DAG-like substances in a voltage-dependent manner (time constant of inactivation decreased by 2- and 3.6-fold at −60, and −30 mV, respectively). In cell-attached patches, treatment with DAG compounds increased the percentage of current traces showing no single Na⁺ channel openings in response to depolarizing voltage-clamp pulses. Moreover, the average of current traces containing single Na⁺ channel openings was essentially the same in control conditions and after treatment with DAG compounds. Removal of Na⁺ channel inactivation by the alkaloid batrachotoxin prevented the attenuation of Na⁺ currents by PKC activation via DAGs. Taken together, these data strongly suggest that PKC-induced attenuation of Na⁺ currents is linked to an enhancement of Na⁺ channel inactivation. This attenuation is caused by an increase in the number of Na⁺ channels inactivating directly from the closed state(s). This inactivation pathway represents a simple and efficient physiological mechanism by which PKC activation might modulate the electrical activity of excitable cells.