The 2-dimensional (2D) color Doppler (2D-CD) proximal isovelocity surface area (PISA) method assumes a hemispheric flow convergence zone to estimate transvalvular flow. Recently developed 3-dimensional (3D)-CD can directly visualize PISA shape and surface area without geometric assumptions. To validate a novel method to directly measure PISA using real-time 3D-CD echocardiography, a circulatory loop with an ultrasound imaging chamber was created to model mitral regurgitation (MR). Thirty-two different regurgitant flow conditions were tested using symmetric and asymmetric flow orifices. Three-dimensional-PISA was reconstructed from a hand-held real-time 3D-CD data set. Regurgitant volume was derived using both 2D-CD and 3D-CD PISA methods, and each was compared against a flow-meter standard. The circulatory loop achieved regurgitant volume within the clinical range of MR (11 to 84 ml). Three-dimensional-PISA geometry reflected the 2D geometry of the regurgitant orifice. Correlation between the 2D-PISA method regurgitant volume and actual regurgitant volume was significant (r(2) = 0.47, p <0.001). Mean 2D-PISA regurgitant volume underestimate was 19.1 +/- 25 ml (2 SDs). For the 3D-PISA method, correlation with actual regurgitant volume was significant (r(2) = 0.92, p <0.001), with a mean regurgitant volume underestimate of 2.7 +/- 10 ml (2 SDs). The 3D-PISA method showed less regurgitant volume underestimation for all orifice shapes and regurgitant volumes tested. In conclusion, in an in vitro model of MR, 3D-CD was used to directly measure PISA without geometric assumption. Compared with conventional 2D-PISA, regurgitant volume was more accurate when derived from 3D-PISA across symmetric and asymmetric orifices within a broad range of hemodynamic flow conditions.