Lung ventilation was mapped in seven healthy male Sprague-Dawley rats (433 ± 24 g) using hyperpolarized 129Xe magnetic resonance imaging (MRI) at 3.0 T, and validated with hyperpolarized 3He MRI under similar ventilator conditions. Ventilation maps were obtained using flip angle variation for offset of RF and relaxation (FAVOR) which is a multiple breath imaging technique that extracts the fractional ventilation parameter, r, on a pixel-by-pixel basis from the dynamic signal enhancement. r is defined as the fractional refreshment of gas per breath. Under the ventilator conditions used in this work, whole-lung measurements of fractional ventilation obtained using hyperpolarized 129Xe were not significantly different from those obtained using hyperpolarized 3He (p = 0.8125 by a Wilcoxon matched pairs test). Fractional ventilation gradients calculated in the superior/inferior (S/I) and anterior/posterior (A/P) directions obtained using hyperpolarized 129Xe were not significantly different from those obtained using hyperpolarized 3He (p = 0.9375 and p = 0.1563, for the S/I and A/P directions, respectively). Following baseline fractional ventilation measurements, one representative rat was challenged with methacholine and fractional ventilation measurements were performed over a time course of 10 min. A reduction and subsequent recovery in whole-lung r values were detected using the FAVOR method. Magn Reson Med, 2012. © 2012 Wiley Periodicals, Inc.