Background Assessment of the left ventricular (LV) and the right ventricular (RV) volumes and their functions is important for prognostic prediction and clinical decision making. We compared the accuracy for quantifying the LV and the RV volumes in vitro between conventional two-dimensional echocardiography (2DE) and real-time three-dimensional echocardiography (RT3DE). Methods The volumes of 37 rubber-models (10 regularly shaped to simulate normal LV, 7 shaped to simulate LV with symmetric aneurysm, 8 shaped to simulate LV with asymmetric aneurysm, and 12 irregularly shaped to simulate normal RV) and 10 excised canine hearts were measured by RT3DE and 2DE. On RT3DE full volume imaging, the inner-surfaces of the rubber-models and canine LV and RV were outlined and the volumes were measured using 2-, 4-, 8- and 16-plane methods with the RT3DE analysis software. On 2DE imaging, the volumes were measured by the Simpson method. The LV and RV volumes measured by drained water were served as reference values, with which we compared RT3DE and 2DE data.Results In rubber models mimicking normal LV and LV with symmetric aneurysms, RT3DE results were strongly correlated with reference values (r=0.795-0.998) and there was a good correlation between 2DE estimates and reference values (r=0.715-0.729). There were no significant differences between RT3DE estimates, 2DE results and reference values (P>0.05). In rubber models mimicking the RV and LV with asymmetric aneurysm, RT3DE strongly correlated with reference values (r=0.765-0.988), but 2DE weakly correlated with reference values (r=0.518-0.592). There were no differences between RT3DE and reference values (P>0.05), but a significant difference between 2DE and reference values occurred (P<0.05). For excised canine hearts, there was a strong correlation between RT3DE and reference values (r=0.728-0.914), while 2DE showed a less obvious correlation (r=0.502-0.615). Again, there were no significant differences between RT3DE and reference values (P>0.05), but there was a significant difference between 2DE and reference values (P<0.05).Conclusions RT3DE can accurately quantify LV and RV volumes and provides a new tool to evaluate LV and RV function. For LV and RV measurements by RT3DE, 8-plane strategy is the optimum choice for accuracy and convenience.