To calculate left ventricular (LV) volume by two-dimensional echocardiography (2DE), assumptions must be made about ventricular symmetry and geometry. Three-dimensional echocardiography (3DE) can quantitate LV volume without these limitations, yet its incremental value over 2DE is unknown. The purpose of this study was to compare the accuracy of LV volume determination by 3DE to standard 2DE methods. To compare the accuracy of 3DE with standard 2DE algorithms for quantitating LV volume, 28 excised canine ventricles of known volume and varying shapes (15 symmetric and 13 aneurysmal) and 10 instrumented dogs prepared so that instantaneous ventricular volume could be measured were examined by 2DE (bullet and biplane Simpson's formulas) and again by 3DE. In both excised and beating hearts, 3DE was more accurate in quantitating volume than either 2DE method (excised: error = 0.6 ± 3.2, 2.5 ± 10.7, and 4.0 ± 8.5 ml by 3D, bullet, and Simpson's, respectively; beating: error = −0.5 ± 3.5, −0.3 ± 9.6, and −7.6 ± 8.0 ml by 3DE, bullet, and Simpson's, respectively). This difference in accuracy between 3DE and 2DE methods was especially apparent in asymmetric ventricles distorted by ischemia or right ventricular volume overload. Stroke volume and ejection fraction calculated by 3DE also demonstrated better agreement with actual values than the bullet or Simpson methods with less variability (ejection fraction: error = −0.2% ± 5.1%, 7.7% ± 8.5%, and 6.8% ± 12.3% by 3DE, bullet, and Simpson's, respectively). In both in vitro and in vivo settings, 3DE provides improved accuracy for LV volume and performance than current 2DE algorithms.