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Three-dimensional cardiac activation imaging (3-DCAI) aims at imaging the activation sequence throughout the 3-D myocardium. In the present study, the performance of 3-DCAI was validated through both in vivo animal experiments and computer simulations under a pacing protocol. The non-invasively imaged activation sequence from body surface potential maps (BSPMs) was quantitatively compared with the measured activation sequence obtained from the simultaneous intramural recording using a 3-D intra-cardiac mapping technique in a rabbit model. In addition, computer simulations were conducted to provide further assessment of the performance of the 3-DCAI algorithm in a realistic-geometry rabbit heart-torso model. The encouraging results suggest that 3-DCAI can non-invasively image the activation sequence and localize the origin of activation with good accuracy.