The cardiac Ca²⁺-independent transient outward K⁺ current (I_to), a major repolarizing ionic current, is markedly affected by Cl^- substitution and anion channel blockers. We re-explored the mechanism of the action of anions on I_to by using whole-cell patch-clamp in single isolated rat cardiac ventricular myocytes. The transient outward current was sensitive to blockade by the K⁺ channel blocker 4-aminopyridine (4-AP) and was abolished by Cs⁺ substitution for intracellular K⁺. Equimolar replacement of most of the extracellular Cl^- with less permeant anions, aspartate (Asp^-) and glutamate (Glu^-), markedly suppressed the current. Removal of external Na⁺ or pretreatment of myocytes with phalloidin, a stabilizer of F-actin, did not significantly affect the inhibitory action of less permeant anions on I_to. In contrast, the permeant Cl^- substitute Br^- did not markedly affect the current, whereas F^- substitution for Cl^- induced a slight inhibition. The I_to elicited during Br^- substitution for Cl^- was also sensitive to blockade by 4-AP. The ability of different anions to shift the steady-state inactivation curve of I_to from a more negative potential to a more positive potential was in the sequence: NO₃^- > Cl^- ~ Br^- > Gluconate^- > Glu^- > Asp^-. Depolymerization of actin filaments with cytochalasin D (CytD) induced an effect on the steady-state inactivation of I_to similar to that of less permeant anions. Fluorescent phalloidin staining experiments revealed that CytD-pretreatment significantly decreased the intensity of FITC-phalloidin staining of F^- actin, whereas Asp^--substitution for Cl^- was without significant effect on the intensity. These results suggest that the I_to channel is modulated by anion channel(s), in which the actin cytoskeleton may be implicated.