BACKGROUND: Strain and strain rate have been proposed as tools to quantify regional myocardial function. One of the major pitfalls of the current methodology is its angle dependency. To overcome this problem, we have developed a new method for the estimation of strain, independent of angle. The aim of this study was to validate this new methodology in an experimental setting using sonomicrometry. METHODS AND RESULTS: In 5 open-chest sheep, ultrasound data were acquired. The new methodology was used to perform simultaneous measurements of radial and longitudinal strain in the inferolateral wall. Segment-length sonomicrometry crystals were used as the reference. After baseline acquisitions, deformation was modulated by pharmacologically changing the inotropic state of the myocardium and by inducing ischemia. Ultrasonically estimated radial and longitudinal strain were validated against sonomicrometry by means of Bland-Altman analysis and the intraclass correlation coefficient. For both strain components, good agreements were found between the ultrasound and the sonomicrometry measurements as shown by Bland-Altman statistics. The intraclass correlation coefficients were found to be 0.72 and 0.80 for the radial and longitudinal components, respectively. CONCLUSIONS: A new technique for the estimation of myocardial deformation was validated. It was shown that the current problem of angle dependency was solved and that 2 deformation components could be estimated simultaneously and accurately. Furthermore, the technique was less time-consuming, because anatomic tracking was performed automatically. This approach could potentially accelerate the clinical acceptance of ultrasound deformation imaging in cardiology.