Recently a new acoustic-quantification (AQ) technique has been developed to provide on-line automated border detection with an integrated backscatter analysis. Prior studies have largely correlated AQ areas with volumes without direct comparison of volumes for agreement. By using complete AQ-detected borders as the input to a validated method for three-dimensional echocardiographic (3DE) reconstruction, we can compare an entire cavity volume measured with the aid of AQ against a directly measured volume. This would also explore the possibility of applying AQ to 3DE reconstruction to reduce tracing time and enhance routine applicability. To compare reconstructed volumes with actual values in a stable standard allowing direct volume measurement, the left ventricles of 13 excised animal hearts were studied with a 3DE system that automatically combines two-dimensional (2D) images and their locations. Intersecting 2D views were obtained with conventional scanning and AQ imaging, with gains optimized to permit 3D reconstruction by detecting the most continuous AQ borders for each view, with maximal cavity size. Reconstruction was performed with manually traced central endocardial reflections and AQ-detected borders. 3DE reconstructions of both manually and AQ-detected borders visually reproduced the left ventricular shapes; the AQ reconstructions, however, were consistently smaller. The reconstructed left ventricular (LV) volumes correlated well with actual values by both manual and AQ techniques (r = 0.93 and 0.88, with standard errors of 2.3 cc and 2.0 cc, p = not significant [NS]). Agreement with actual values was relatively close for the manually traced borders (y = 0.93x + 0.68, mean difference = −0.8 ± 2.2 cc). AQ-derived reconstructions consistently underestimated LV volume by 39 ± 10% (y = 0.62x − 0.09, mean difference = −7.8 ± 3.0 cc, different from manually traced and actual volumes by analysis of variance [ANOVA], F = 69, p < 0.00001). The AQ-detected threshold signal was displaced into the cavity, and volume between walls and false tendons was excluded, leading to underestimation, which increased with increasing cavity volume (r = 0.76). The AQ technique can therefore be applied to 3DE reconstruction, providing volumes that correlate well with directly measured values in a stable in vitro standard, minimizing observer decisions regarding manual border placement after image acquisition. However, when the complete borders needed for 3D reconstruction are used, absolute volumes are underestimated with current algorithms that integrate backscatter and displace the detected threshold into the ventricular cavity.