Background: The effects of hemodialysis (HD) on left ventricular (LV) function have been studied by various echocardiographic techniques (M-mode, 2D echocardiography). These studies are hampered by a low accuracy of measurements because of geometric assumptions regarding LV shape. Three-dimensional echocardiography (3DE) overcomes this limitation. Methods: We tested the feasibility of 3DE assessment of LV function during HD. Conventional biplane Simpson rule (BSR) and single plane area length method (SPM) for LV function analysis were used as a reference. Results: 12 HD patients were studied and in 10 (83%) a total of 80 3D datasets were acquired. In 3 patients, one dataset (4%) was of insufficient quality and excluded from analysis. Correlation between SPM, BSR and 3DE for calculation of end-diastolic (EDV, r = 0.89 and r = 0.92, respectively), end-systolic volume (ESV, r = 0.92 and r = 0.93, respectively) and for ejection fraction (EF, r = 0.90 and r = 0.88, respectively) was moderate. Limits-of-agreement results for EDV and ESV were poor with confidence intervals larger than 30 ml. Both 2DE methods underestimated end-diastolic and end-systolic volume, while overestimating ejection fraction. Conclusion: 3DE is feasible for image acquisition during HD, which opens the possibility for accurate and reproducible measurement of LV function during HD. This may improve the assessment of the acute effect of HD on LV performance, and guide therapeutic strategies aimed at preventing intradialytic hypotension.