Despite the potential of real-time three-dimensional (3D) echocardiography (RT3DE) to assess myocardial perfusion, there is no quantification method available for perfusion analysis from RT3DE images. Such method would require 3D regions of interest (ROI) to be defined and adjusted frame-by-frame to compensate for cardiac translation and deformation. Our aims were to develop and test a technique for automated identification of 3D myocardial ROI suitable for translation-free quantification of myocardial videointensity over time, MVI(t), from contrast-enhanced RT3DE images. Twelve transthoracic RT3DE (Philips) data sets obtained in pigs during transition from no contrast to steady-state enhancement (Definity) were analysed using custom software. Analysis included: (i) semi-automated detection of left ventricular endo- and epicardial surfaces using level-set techniques in one frame to define a 3D myocardial ROI, (ii) rigid 3D registration to reduce translation and rotation, (iii) elastic 3D registration to compensate for deformation, and (iv) quantification of MVI(t) in the 3D ROI from the registered and non-registered data sets to assess the effectiveness of registration. For each MVI(t) curve we computed % variability during steady-state enhancement (100 x SD/mean) and goodness of fit (r2) to the indicator dilution equation MVI(t) = A[1-exp(-betat)]. Analysis of myocardial contrast throughout contrast inflow was feasible in all data sets. Three-dimensional registration improved MVI(t) curves in terms of both % variability (2.8 +/- 1.8 to 1.5 +/- 0.9%; P < 0.05) and goodness of fit (r2 from 0.79 +/- 0.2 to 0.90 +/- 0.1; P < 0.05). This is the first study to describe a new technique for semi-automated volumetric quantification of myocardial contrast from RT3DE images that includes registration and thus provides the basis for 3D measurement of myocardial perfusion.