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Although 2D speckle tracking echocardiography (STE) has been shown useful for the assessment of regional left ventricular (LV) function, it is limited by its 2D nature. Our goal was to evaluate new 3D-STE software by comparing regional wall motion (RWM) measurements against 2D-STE, and testing its ability to identify RWM abnormalities. 2D and real-time 3D datasets obtained in 32 subjects were analyzed to measure radial, longitudinal and rotational indices of displacement and strain. Segments were classified as normal or abnormal using cardiac MRI. 3D-STE and 2D-STE indices did not correlate well and showed wide limits of inter-technique agreement despite the minimal biases. In normal segments, 3D-STE showed: (1) higher displacements, due the out-of-plane motion component; (2) smaller SDs, indicating tighter normal ranges; (3) gradual decrease in displacement and reversal in rotation from base to apex. In abnormal segments, all 3D-STE indices were reduced. In conclusion, this is the first study to evaluate the new 3D-STE technique for measurements of RWM indices and demonstrate its superiority over 2D-STE.