Purpose-To investigate immobilization-induced ventilation defects when performing hyperpolarized 3 He (H 3 He) MRI of the lung.Methods and Materials-Twelve healthy subjects underwent MRI of the lungs following inhalation of H 3 He gas at three time points: 1) immediately after having been positioned supine on the MR scanner table, 2) at 45 minutes while remaining supine, 3) and immediately thereafter after having turned prone. All image sets were reviewed in random order by three independent, blinded readers who recorded number, location and size of H 3 He ventilation defects. Scores were averaged for each time point and comparisons were made to determine change in number, location and size of ventilation defects with time and positioning of the subject in the scanner.Results-At baseline supine there were small numbers of defects in the dependent (posterior) and non-dependent (anterior) portions of the lung (p=0.625). At 45 minutes there was a significant increase in the mean number of ventilation defects/slice (VDS) for the dependent (p=0.005) and a decrease for the non-dependent lung portions (p=0.021). After subjects turned prone, mean VDS for posterior defects decreased significantly (p=0.011) while those for anterior defects increased (p=0.010). Most defects were less than 3 cm in diameter.Conclusion-It was found that immobilization of the subject for an extended period of time led to increased number of H 3 He ventilation defects in the dependent portions of the lung. Therefore, after a subject is positioned in the scanner, H 3 He MR imaging should be performed quickly to avoid the occurrence of the immobilization-induced ventilation defects, and possible overestimation of disease.