Three-dimensional (3-D) cardiac activation imaging (3-DCAI) is a recently developed technique that aims at imaging the activation sequence throughout the 3-D volume of myocardium. 3-DCAI entails the modeling and estimation of the cardiac equivalent current density (ECD) distribution from which the local activation time within myocardium is determined as the time point with the peak amplitude of local ECD estimates. In this paper, we report, for the first time, an experimental study of the performance and applicability of 3-DCAI as judged by measured 3-D cardiac activation sequence using 3-D intra-cardiac mapping, in a group of 4 healthy rabbits during ventricular pacing. During the experiments, the body surface potentials and the intramural bipolar electrical recordings were simultaneously measured in a closed-chest condition to allow for a rigorous evaluation of the noninvasive 3-DCAI algorithm using the intra-cardiac mapping. The ventricular activation sequence non-invasively imaged from the body surface measurements by using 3-DCAI was generally in agreement with that obtained from the invasive intra-cardiac recordings. The overall difference between them, quantified as the root mean square (RMS) error, was 7.42±0.61 ms, and the normalized difference, quantified as the relative error (RE), was 0.24±0.03. The distance from the reconstructed site of initial activation to the actual pacing site, defined as the localization error (LE), was 5.47±1.57 mm. In addition, computer simulations were conducted to provide additional assessment of the performance of the 3-DCAI algorithm using a realistic-geometry rabbit heart-torso model. Averaged over 9 pacing sites, the RE and LE were 0.20±0.07 and 4.56±1.12 mm, respectively, for single-pacing, when 20 μV Gaussian white noise was added to the body surface potentials at 53 body surface locations. Averaged over 8 pairs of dual pacing, the RE was 0.25±0.06 for 20 μV additive noise. The present results obtained through both in vivo experimentation and computer simulation suggest that 3-DCAI can non-invasively capture important features of ventricular excitation (e.g. the activation origin and the activation sequence), and has the potential of becoming a useful imaging tool aiding cardiovascular research and clinical diagnosis and management of cardiac diseases.