Background— It has been demonstrated that spironolactone (SP) decreases the QT dispersion in chronic heart failure. In this study, the effects of SP and its metabolite, canrenoic acid (CA), on human ether-a-go-go–related gene (HERG) currents were analyzed. Methods and Results— HERG currents elicited in stably transfected Chinese hamster ovary cells were measured with the whole-cell patch-clamp technique. SP decreased HERG currents in a concentration-dependent manner (IC ₅₀ =23.0±1.5 μmol/L) and shifted the midpoint of the activation curve to more negative potentials (Vh=−13.1±3.4 versus −18.9±3.6 mV, P <0.05) without modifying the activation and deactivation kinetics. SP-induced block (1 μmol/L) appeared at the range of membrane potentials coinciding with that of channel activation, and thereafter, it remained constant, reaching 24.7±3.8% at +60 mV (n=6, P <0.05). CA (0.01 nmol/L to 500 μmol/L) blocked HERG channels in a voltage- and frequency-independent manner. CA at 1 nmol/L shifted the midpoint of the activation curve to −19.9±1.8 mV and accelerated the time course of channel activation (τ=1064±125 versus 820±93 ms, n=11, P <0.01). The envelope of the tail test demonstrated that at the very beginning of the pulses to +40 mV (25 ms), a certain amount of block was apparent (31.3±9.9%). CA did not modify the voltage-dependence of HERG channel inactivation (Vh=−60.8±5.6 versus −62.9±3.1 mV, n=6, P >0.05) or the kinetics of the reactivation process at any potential tested. CA and aldosterone also blocked the native I _Kr in guinea-pig ventricular myocytes. Conclusions— At concentrations reached after administration of therapeutic doses of SP, CA blocked the HERG channels by binding to both the closed and open states of the channel.