A hyperpolarized 3He ventilation imaging protocol was implemented to assess alveolarpO2 values and the oxygen depletion rate in rats. The imaging protocol, which is based on spiralk-space sampling, was designed to acquire a high signal-to-noise ratio (SNR)T1-weighted ventilation series of images in a single breath-hold. Simulations were performed to estimate the accuracy and dependence of thepO2 imaging protocol on the image SNR and the RF flip-angle determination. The imaging protocol was validated in vitro in phantoms and in vivo in rats. Imaging sessions were carried out for different inhaled O2 concentrations ranging from 20% to 40%. Parametric maps of alveolarpO2 and oxygen depletion rate were generated from the series of images. For each investigated animal, the differences in measured alveolarpO2 values are in agreement with the changes in inhaled O2 concentration. The oxygen depletion rates, ranging between 0.7 and 8.0 mbar s−1, are in close agreement with the published values for healthy rats. Magn Reson Med, 2007. © 2006 Wiley-Liss, Inc.