Background. Current two-dimensional quantitative echocardiographic methods of volume assessment require image acquisition from standardized scanning planes. Left ventricular volume and ejection fraction are then calculated by assuming ventricular symmetry and geometry. These assumptions may not be valid in distorted ventricles. Three-dimensional echocardiography can quantify left ventricular volume without the limitations imposed by the assumptions of two-dimensional methods. We have developed a three-dimensional system that automatically integrates two-dimensional echocardiographic images and their positions in real time and calculates left ventricular volume directly from traced endocardial contours without geometric assumptions. Methods and Results. To study the accuracy of this method in quantifying left ventricular volume and performance in vivo, a canine model was developed in which instantaneous left ventricular volume can be measured directly with an intracavitary balloon connected to an external column. Ten dogs were studied at 84 different cavity volumes (4 to 85 cm3) and in conditions of altered left ventricular shape produced by either coronary occlusion or right ventricular volume overload. To demonstrate clinical feasibility, 19 adult human subjects were then studied by this method for quantification of stroke volume. Left ventricular volume, stroke volume, and ejection fraction calculated by three-dimensional echocardiography correlated well with directly measured values (r=.98, .96, .96 for volume, stroke volume, and ejection fraction, respectively) and agreed closely with them (mean difference, -0.78 cm3, -0.60 cm3, -0.32%). In humans, there was a good correlation (r=.94, SEE=4.29 cm3) and agreement (mean difference, -0.98+/-4.2 cm3) between three-dimensional echocardiography and Doppler-derived stroke volumes. Conclusions. Three-dimensional echocardiography allows accurate assessment of left ventricular volume and systolic function.