Voltage-dependent potassium (Kv) channel trafficking and localization are regulated by proteins of the cytoskeleton, but the mechanisms by which this occurs are still unclear. Using human embryonic kidney (HEK) cells as a heterologous expression system, we have tested the role of the actin cytoskeleton in modulating the function of Kv4.2 channels. Pretreatment (1 hour) of HEK cells with 5 µM cytochalasin D to disrupt the actin microfilaments greatly augments whole cell Kv4.2 currents at potentials positive to -20 mV. However, no changes in the voltage-dependence of activation and inactivation of macroscopic currents were observed to account for this increase. Similarly, single channel recordings failed to reveal any significant changes in the single channel conductance, open probability and kinetics. However, the mean patch current was increased from 0.9 ± 0.2 pA in control to 6.7 ± 3.0 pA in the presence of cytochalasin D. Imaging experiments revealed a clear increase in the surface expression of the channels, and the appearance of "bright spot" features, suggesting that large numbers of channels were being grouped at specific sites. Our data provide clear evidence that increased numbers and altered distribution of Kv4.2 channels at the cell surface are primarily the result of reorganization of the actin cytoskeleton.