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Pennation angle is an important architecture parameter to understand muscle functioning. It is commonly measured using 2D ultrasound. However, it is difficult to infer 3D muscle architecture from 2D imaging. Therefore, we compare the pennation angle measurements obtained with 3D-DTI fiber-tractography and 3D-ultrasound (3D-US). We acquired data of the Vastus Lateralis muscle in 9 healthy subjects. The mean pennation angle with 3D-US was 18.9±5.9, whereas we found 33.3±6.7 (straight fiber approximation) and 34.5±4.8 (curved fiber fit) for DTI fiber-tractography. These differences between 3D-US and DTI could be of technical or physiological origin.