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Background: Planimetry measured by two-dimensional transthoracic echocardiography (TTE, MVA2D) is the reference method for the evaluation of the severity of mitral stenosis (MS) but requires experienced operators and good echocardiographic windows. Real-time three-dimensional transesophageal echocardiography (3D-TEE, MVA3D) may overcome these limitations but its accuracy has never been evaluated.Methods: We prospectively enrolled 80 patients (58±15 years, 86% female) referred for MS evaluation who underwent within one week a clinically indicated TTE and 3D-TEE. MVA2D was measured by experienced operators (Level III), MVA3D by one experienced and one non-experienced (Level I) operators blinded of any clinical or TTE information.Results: MVA3D measured by the experienced operator (1.11±0.32 cm2, median 1.1cm2, range [0.45-2.20]) did not differ from and correlated well with MVA2D (1.10±0.34 cm2, median 1.05cm2, range [0.45-2.30]), p=0.87, r=0.79, p<0.0001) and mean difference between methods was small (+0.004±0.21 cm2). MVA3D measured by the non-experienced operator (1.08±0.34 cm2, median 1.02 cm2; range [0.45-2.23]) also did not differ from and correlated well with MVA2D measured by experienced operators (p=0.25; r=0.86, p<0.0001; mean difference -0.02±0.18 cm2). Intra and interobserver variability was 0.02±0.25cm2 and 0.01±0.33cm2.Conclusion: 3D-TEE provides accurate and reproducible MVA measurements similar to 2D planimetry performed by experienced operators. Thus, 3D-TEE could be considered as an alternative tool for the evaluation of MS severity, especially in patients with poor echocardiographic windows or for non-experienced operators.