Temporary sequential biventricular pacing (BiVP) is a promising treatment for postoperative cardiac dysfunction, but the mechanism for improvement in right ventricular (RV) dysfunction is not understood. In the present study, cardiac output (CO) was optimized by sequential BiVP in six anesthetized, open-chest pigs during control and acute RV pressure overload (RVPO). Ventricular contractility was assessed by the maximum rate of increase of ventricular pressure (dP/dt(max)). Mechanical interventricular synchrony was measured by the area of the normalized RV-left ventricular (LV) pressure diagram (A(PP)). Positive A(PP) indicates RV pressure preceding LV pressure, whereas zero indicates complete synchrony. In the control state, CO was maximized with nearly simultaneous stimulation of the RV and LV, which increased RV (P = 0.006) and LV dP/dt(max) (P = 0.002). During RVPO, CO was maximized with RV-first pacing, which increased RV dP/dt(max) (P = 0.007), but did not affect LV dP/dt(max), and decreased the left-to-right, end-diastolic pressure gradient (P = 0.023). Percent increase of RV dP/dt(max) was greater than LV dP/dt(max) (P = 0.014). There were no increases in end-diastolic pressure to account for increases in dP/dt(max). In control and RVPO, RV dP/dt(max) was linearly related to A(PP) (r = 0.779, P < 0.001). The relation of CO to A(PP) was curvilinear, with a peak in CO with positive A(PP) in the control state (P = 0.004) and with A(PP) approaching zero during RVPO (P = 0.001). These observations imply that, in our model, BiVP optimization improves CO by augmenting RV contractility. This is mediated by changes in mechanical interventricular synchrony. Afterload increases during RVPO exaggerate this effect, making CO critically dependent on simultaneous pressure generation in the RV and LV, with support of RV contractility by transmission of LV pressure across the interventricular septum.