Elevation of external [K +] potentiates outward K + current through several voltage-gated K + channels. This increase in current magnitude is paradoxical in that it occurs despite a significant decrease in driving force. We have investigated the mechanisms involved in K +-dependent current potentiation in the Kv2.1 K + channel. With holding potentials of −120 to −150 mV, which completely removed channels from the voltage-sensitive inactivated state, elevation of external [K +] up to 10 mM produced a concentration-dependent increase in outward current magnitude. In the absence of inactivation, currents were maximally potentiated by 38%. At more positive holding potentials, which produced steady-state inactivation, K +-dependent potentiation was enhanced. The additional K +-dependent potentiation (above 38%) at more positive holding potentials was precisely equal to a K +-dependent reduction in steady-state inactivation. Mutation of two lysine residues in the outer vestibule of Kv2.1 (K356 and K382), to smaller, uncharged residues (glycine and valine, respectively), completely abolished K +-dependent potentiation that was not associated with inactivation. These mutations did not influence steady-state inactivation or the K +-dependent potentiation due to reduction in steady-state inactivation. These results demonstrate that K +-dependent potentiation can be completely accounted for by two independent mechanisms: one that involved the outer vestibule lysines and one that involved K +-dependent removal of channels from the inactivated state. Previous studies demonstrated that the outer vestibule of Kv2.1 can be in at least two conformations, depending on the occupancy of the selectivity filter by K + (Immke, D., M. Wood, L. Kiss, and S. J. Korn. 1999. J. Gen. Physiol. 113:819–836; Immke, D., and S. J. Korn. 2000. J. Gen. Physiol. 115:509–518). This change in conformation was functionally defined by a change in TEA sensitivity. Similar to the K +-dependent change in TEA sensitivity, the lysine-dependent potentiation depended primarily (>90%) on Lys-356 and was enhanced by lowering initial K + occupancy of the pore. Furthermore, the K +-dependent changes in current magnitude and TEA sensitivity were highly correlated. These results suggest that the previously described K +-dependent change in outer vestibule conformation underlies the lysine-sensitive, K +-dependent potentiation mechanism.