Three-dimensional (3D) strain analysis based on real-time 3-D echocardiography (RT3DE) has emerged as a novel technique to quantify regional myocardial function. The goal of this study was to evaluate accuracy of a novel model-based 3D tracking tool (eSie Volume Mechanics, Siemens Ultrasound, Mountain View, CA, USA) using sonomicrometry as an independent measure of cardiac deformation. Thirteen sheep were instrumented with microcrystals sutured to the epi- and endocardium of the inferolateral left ventricular wall to trace myocardial deformation along its three directional axes of motion. Paired acquisitions of RT3DE and sonomicrometry were made at baseline, during inotropic modulation and during myocardial ischemia. Accuracy of 3D strain measurements was quantified and expressed as level of agreement with sonomicrometry using linear regression and Bland-Altman analysis. Correlations between 3D strain analysis and sonomicrometry were good for longitudinal and circumferential strain components (r = 0.78 and r = 0.71) but poor for radial strain (r = 0.30). Accordingly, agreement (bias +/- 2SD) was -5 +/- 6 % for longitudinal, -5 +/- 7 % for circumferential, and 15 +/- 19 % for radial strain. Intra-observer variability was low for all components (intra-class correlation coefficients (ICC) of respectively 0.89, 0.88 and 0.95) while inter-observer variability was higher, in particular for radial strain (ICC = 0.41). The present study shows that 3D strain analysis provided good estimates of circumferential and longitudinal strain, while estimates of radial strain were less accurate between observers.