Background: The slope of the action potential duration (APD) restitution curve may be a significant determinant of the propensity to develop ventricular fibrillation, with steeper slopes associated with a more arrhythmogenic substrate. We hypothesized that one mechanism by which beta-blockers reduce sudden cardiac death is by flattening the APD restitution curve. Therefore, we investigated whether infusion of esmolol modulates the APD restitution curve in vivo. In 10 Yorkshire pigs, dynamic APD restitution curves were determined from measurements of APD₉₀ by a monophasic action potential catheter positioned against the right ventricular septum during right ventricular apical pacing in the basal state and during infusion of esmolol. APD restitution curves were fitted to the 3 parameter exponential equation, APD = a*(1-e^(-b*DI)) + c, where DI is the diastolic interval. Esmolol decreased the maximal APD slope, 0.68 0.14 vs. 0.94 ± 0.24 (baseline), p = 0.002, and flattened the APD restitution curve at shorter DIs, 75 and 100 ms (p < 0.05). To compare the slopes of the APD restitution curves at similar steady states, slopes were also computed at points of intersection between the restitution curve and the lines representing pacing at a fixed cycle length (CL) of 200, 225, 250, 275, and 300 ms using the relationship CL = APD + DI. Esmolol decreased APD restitution slopes at CLs 200 to 275 ms (p < 0.05). Esmolol flattens the cardiac APD restitution curve in vivo, particularly at shorter cycle lengths and diastolic intervals. This may represent a novel mechanism by which betablockers prevent sudden cardiac death.