The objective of this study was to validate a real-time 3-dimensional echocardiography (3DE) technique for the determination of left ventricular (LV) volume and ejection fraction (EF). In 10 mongrel dogs, an electromagnetic flow (EMF) probe was placed on the aorta, and the thorax was closed. Transthoracic imaging was performed during multiple hemodynamic conditions (n = 58) with simultaneous measurement of stroke volume (SV) with the use of EMF. From the volumetric data set, LV volumes were manually traced off-line by 2 independent observers with an apical rotation method (6 planes) and a conventional method (biplane) in a subset of conditions. This tracing technique was also evaluated in 18 human subjects in whom the calculated EF values were compared with values derived by multigated radionuclide angiography (MUGA). Excellent correlation and close limits of agreement were noted between SV measured by 3DE and EMF (r = 0.93) in dogs. In comparison with EMF-derived SV, 3DE provided better correlation than the biplane method (r = 0.93 versus r = 0.61). Interobserver and intraobserver variabilities were comparable (r = 0.94 and r = 0.94, respectively). In a comparison of MUGA-derived EF values and those obtained by 3DE in human subjects, 3DE provided better correlation than the biplane method (r = 0.94 versus r = 0.85). Real-time 3DE accurately measures left ventricular volumes transthoracically over a wide range of hemodynamic conditions in dogs and accurately determines EF in humans.