Objective: Cardiac repolarization is prolonged and repolarization reserve (RR) is diminished in female rabbits and humans, compared to males. Reduced RR is evidenced by the relatively greater increase in ventricular action potential duration (APD) in myocytes from females in response to drugs that block repolarizing K⁺ currents. Mice are an increasingly important experimental model animal for cardiovascular research, but gender-dependent differences have not been reported for repolarization in murine ventricular myocytes. Methods: APD and repolarizing K⁺ currents were measured in isolated ventricular myocytes from adult littermate male and female mice. Repolarizing K⁺ currents were dissected into transient (I_to) and sustained (I_sus) components and the selective I_sus antagonist FK506 was used to probe for differences in RR. Results: Under control conditions APD at 50% (APD₅₀) and at 90% (APD₉₀) repolarization was significantly longer in females (APD₅₀=15±3 ms, n=6 and APD₉₀=63±6 ms, n=6) compared to males (APD₅₀=8±2 ms, n=7 and APD₉₀=42±9 ms, n=7) at 1.0 Hz. At 0.3 Hz stimulation frequency APD₉₀, but not APD₅₀, was significantly longer in females (APD₅₀=12±2 ms and APD₉₀=54±5 ms, n=10) compared to males (APD₅₀=11±2 ms and APD₉₀=47±7 ms, n=10). FK506 treatment (25 μM) selectively and equally inhibited I_sus in all cells, and significantly increased APD₅₀ and APD₉₀ in males and females at 0.3 and 1.0 Hz. However, increases in APD₅₀ and APD₉₀ (0.3 and 1.0 Hz) in response to FK506 were significantly greater in myocytes from females compared to males. Voltage clamp measurement of I_to and I_sus revealed that males had a relatively more prominent I_to while females exhibited a more prominent I_sus. Conclusions: Ventricular action potential repolarization is prolonged in myocytes from female compared to male mice. Female mice have reduced RR that is unmasked by FK506. These findings suggest that gender is an important variable for cardiovascular studies using mice.