As compared with two-dimensional (2-D) transesophageal echocardiography (TEE), 3-D echocardiography now permits more realistic visualization of cardiac anatomy and of intracardiac lesions. The aim of this study was to apply newer 3-D echocardiographic techniques to quantify volumes of intracardiac masses undergoing surgical resection seen during an intraoperative TEE. The calculated volumes were compared with actual in vitro measurements of surgically resected masses. A total of 14 patients (9 men; 5 women; age range between 21 and 77 years) with intracardiac mass lesions (4 tumors: 3 left atrial myxomas and 1 mitral valve fibroelastoma, and 10 vegetations: 5 aortic valve, 3 mitral valve, 1 tricuspid and 1 pulmonary valve) were studied. Using commercially available 3-D reconstruction software (TomTec v. 4.1), the volumes of intracardiac masses were estimated using both the average rotation (rotation around the long axis, AR) and disk summation (parallel short axis cuts, DS) methods. Volumes of these lesions were also measured in vitro by water submersion. They ranged from 0.20 mL to 24 mL (mean +/- SD = 8.07 +/- 9.21 mL). Both 3-D TEE AR and 3-D TEE DS calculated volumes correlated excellently with in vitro measured volumes (r = 1.00 and r = 0.98, respectively, p = < 0.0001). The correlation between 3-D TEE AR and 3-D TEE DS calculated volumes was also excellent (r = 0.98, p = < 0.0001). In conclusion, the volume assessments by 3-D TEE of intracardiac mass lesions correlated well with in vitro measured volumes of surgical specimens. This technique may prove to be valuable in further defining intracardiac pathology and is a further advancement toward the application of clinically useful 3-D echocardiography.