Twenty-one explanted fixed hearts (14 dogs and 7 pigs) were examined to validate newly developed real-time three-dimensional (RT3D) echocardiography for measurement of left ventricular (LV) mass in vitro and to compare its accuracy and variability with those of conventional echocardiographic measurements. There was an excellent correlation and high degree of agreement for the determination of LV mass between RT3D echocardiography and true mass measurement (r = 0.98; standard error of the estimate [SEE] = 7.3 g; absolute difference [AD] = 2.8 g; y = 1.00 x -4.0, interobserver variability; 5.0%). The conventional echocardiographic methods yielded weaker correlations, larger standard errors, and interobserver variability (area-length method: r = 0.90; SEE = 13.3 g; AD = 13.2 g; 13.3 % / truncated ellipsoid method: r = 0.91; SEE = 14.7 g; AD = 10.5 g; 7. 9% / M-mode: r = 0.91; SEE = 16.2 g; AD = 9.4 g; 15.3%). Determination of LV mass by RT3D echocardiography has a high degree of accuracy and is superior to conventional one- and two-dimensional echocardiographic methods.