Background Zatebradine is a bradycardic agent that inhibits the hyperpolarization-activated current (I f ) in the rabbit sinoatrial node. It also prolongs action potential duration in papillary muscles in guinea pigs and in Purkinje fibers in rabbits. The underlying mechanism by which zatebradine induces this effect has not been explored, but it is likely to involve K + channel block. Methods and Results Cloned human cardiac K + delayed rectifier currents (hKv1.5) were recorded in Ltk − cells transfected with their coding sequence. Zatebradine 10 μmol/L did not modify the initial activation time course of the current but induced a subsequent decline to a lower steady-state current level with a time constant of 109±16 ms. Zatebradine inhibited hKv1.5 with an apparent K D of 1.86±0.14 μmol/L. Block was voltage dependent (electrical distance δ=0.177±0.003) and accumulated in a use-dependent manner during 0.5- and 1-Hz pulse trains because of slower recovery kinetics in the presence of the drug. Zatebradine reduced the tail current amplitude, recorded at −30 mV, and slowed the deactivation time course, which resulted in a “crossover” phenomenon when control and zatebradine tail currents were superimposed. Conclusions These results indicate that (1) zatebradine is an open-channel blocker of hKv1.5, (2) binding occurs in the internal mouth of the ion pore, (3) unbinding is required before the channel can close, and (4) zatebradine-induced block is use dependent because of slower recovery kinetics in the presence of the drug. These effects may explain the prolongation of the cardiac action potential and could be clinically relevant.