The action of AG1478, a potent protein tyrosine kinase (PTK) inhibitor, on rat brain Kv1.5 channels (Kv1.5) stably expressed in Chinese hamster ovary cells was investigated using the whole-cell patch-clamp technique. AG1478 rapidly and reversibly inhibited Kv1.5 currents at +50 mV in a concentration-dependent manner with an IC₅₀ of 9.82 µM. AG1478 accelerated the decay rate of inactivation of Kv1.5 currents without modifying the kinetics of current activation. Pretreatment with the structurally dissimilar PTK inhibitors, genistein and lavendustin A, had no effect on Kv1.5 currents, nor did they modify the AG1478-induced inhibition of Kv1.5. The rate constants for binding and unbinding of AG1478 were 1.46 µM^-1s^-1 and 10.19 s-¹, respectively. AG1478 slowed the deactivation time course, resulting in a tail crossover phenomenon. AG1478-induced inhibition of Kv1.5 channels was voltage-dependent, with a steep increase over the voltage range of channel opening. However, the inhibition exhibited voltage independence over the voltage range in which channels are fully activated. AG1478 induced use-dependent inhibition. The present results suggest that AG1478 acts directly on Kv1.5 as an open-channel blocker, and independently of its effects on PTK activity.