Active stress (˙A) developed by cardiac muscle has been measured in isolated muscle preparations, under physiological loading conditions, by subtracting the passive stress (˙P) from the total stress (˙T). We previously developed a mechanical model based on M-mode ultrasound imaging to calculate these stresses in beating hearts. However, this model was based on one-dimensional imaging information and could not estimate regional differences in ˙A. In the current study this model was improved by including two-dimensional B-mode echocardiographic data. Methods: In a porcine model a micro-manometer tipped catheter was used to measure left-ventricular pressure (LVP) and B-mode ultrasound images were recorded in a short-axis view. On the ultrasound image points in the mid-wall were selected and tracked to completely define the deformation of the myocardium. A kinematic model of the LV was then constructed from the displacement vectors of these points. ˙Twas calculated from the LVP. The material parameters for an exponential stress/strain relation were estimated during the diastolic E-wave when it was assumed that ˙A= 0. These parameters were used to calculate ˙Pduring systole and by subtracting this from ˙T, ˙Awas calculated. Results: The timing and shape of ˙Aprofiles match those obtained from isolated muscle experiments. ˙Awas higher and peaked sooner in the posterior wall than in the anterior wall. Conclusion: Regional active stress estimation is possible in normal beating hearts.