AbstractThe aim of this work was to establish a new, fast, and robust method of flip‐angle calibration for magnetic resonance imaging of hyperpolarized 3He. The method called flip‐angle measurement with magnetization inversion is based on acquiring images from periodically inverted longitudinal magnetization created using the spatial modulation of magnetization technique. By measuring the width of the area where the magnetization was inverted by the spatial modulation of magnetization preparation in phase images, the flip angle can be generated using a simple equation. To validate and establish the limits of the proposed method, flip‐angle measurement with magnetization inversion acquisitions were simulated and applied to proton and hyperpolarized 3He phantoms. Then, the calibration procedure was applied during hyperpolarized 3He magnetic resonance imaging in a healthy volunteer. The advantage of the flip‐angle measurement with magnetization inversion method compared with the conventional method based on the assessment of radiofrequency‐decay is that it is free of errors induced by relaxation due to oxygen, by imperfect excitation slice profile and by any diffusion of 3He into and out of the slice. Another advantage is that it does not require image processing with external software and therefore can be performed using the implemented tools on the magnetic resonance workstation. Magn Reson Med, 2011. © 2010 Wiley‐Liss, Inc.