Background-Single-site ventricular pacing in patients with heart failure, atrial fibrillation, and severe atrioventricular (AV) nodal block risks the generation of discoordinate contraction. Whether altering the site of stimulation can offset this detrimental effect and what role sequential right ventricular-left ventricular (RV-LV) stimulation might play in such patients remain unknown. Methods and Results-Nine subjects with heart failure (ejection fraction, 14% to 30%), atrial fibrillation, and AV block were studied by pressure-volume analysis. Ventricular stimulation was applied to the RV (apex and outflow tract), LV free wall, and biventricular (BiV) at 80 and 120 bpm. BiV improved systolic function more than either site alone (dP/dt max , 810Ϯ83, 924Ϯ98, 983Ϯ102 mm Hg/s for RV, LV, BiV, respectively; PϽ0.05), although LV pacing was significantly better than RV pacing. However, only BiV improved diastolic function (isovolumic relaxation) over RV or LV alone. Similar results were obtained for both heart rates. RV pacing site did not alter the BiV effect, and concomitant stimulation of both RV sites did not improve function over each alone. Finally, varying RV-LV delay revealed optimal responses with simultaneous pacing. Conclusions-Simultaneous BiV pacing acutely enhances both systolic and diastolic function over single-site RV or LV pacing in congestive heart failure patients with atrial fibrillation and advanced AV block. Sequential RV-LV stimulation offers minimal benefit on average and should perhaps be considered only in targeted subsets such as nonresponding patients.