Accurately quantifying transmitral flow volume is clinically important not only as a measure of cardiac output, but also as a value from which to subtract aortic flow, for determining the severity of mitral regurgitation. However, controversy exists over the accuracy of pulsed Doppler for mitral flow quantification because of the complexity of mitral flow geometry and dynamic changes in flow profile and flow area. To explore the feasibility of directly quantifying transmitral flow volume with a newly developed dynamic 3-dimensional digital color Doppler technique, this in vivo experimental study was conducted to validate the method. Eight open chest sheep were imaged with a multiplane transesophageal (TEE) probe placed on the heart for digital 3-dimensional gated acquisition of mitral inflow over a 180-degree acquisition. The digital velocity data were contour detected for flow area after computing the velocity vectors and flow profile perpendicular to a spherical 3-dimensional surface across the mitral annulus. Flow areas and actual velocities were then integrated in time and space and the resulting flow volumes were compared with those obtained by a reference electromagnetic flowmeter on the aorta for 26 steady hemodynamic states. The flow volumes correlated closely to the electromagnetic references (y = 0.87x + 2.49, r = 0.92, SEE = 1.9 Ml per beat). Our study shows that transmitral flow volume can be accurately determined in vivo by this dynamic 3-dimensional digital color Doppler flow quantification method.