The goal of this study was to analyze the effects of genistein, a widely used tyrosine kinase inhibitor, on cloned Shaw-type K⁢ currents, Kv3.1 which were stably expressed in Chinese hamster ovary (CHO) cells, using the whole-cell configuration of patch-clamp techniques. In whole-cell recordings, genistein at external concentrations from 10 to 100μM accelerated the rate of inactivation of Kv3.1 currents, thereby concentration-dependently reducing the current at the end of depolarizing pulse with an IC50 value of 15.71⁑0.67μM and a Hill coefficient of 3.28⁑0.35 (n=5). The time constant of activation at a 300 ms depolarizing test pulses from ⁣80 mV to ⁢40 mV was 1.01⁑0.04 ms and 0.90⁑0.05 ms (n=9) under control conditions and in the presence of 20μM genistein, respectively, indicating that the activation kinetics was not significantly modified by genistein. Genistein (20μM) slowed the deactivation of the tail current elicited upon repolarization to ⁣40 mV, thus inducing a crossover phenomenon. These results suggest that drug unbinding is required before Kv3.1 channels can close. Genistein-induced block was voltage-dependent, increasing in the voltage range (⁣20 mV∼0 mV) for channel opening, suggesting an open channel interaction. Genistein (20μM) produced use-dependent block of Kv3.1 at a stimulation frequency of 1 Hz. The voltage dependence of steady-state inactivation of Kv3.1 was not changed by 20μM genistein. Our results indicate that genistein blocks directly Kv3.1 currents in concentration-, voltage-, time-dependent manners and the action of genistein on Kv3.1 is independent of tyrosine kinase inhibition.