Chemical exchange saturation transfer (CEST) is a contrast mechanism that exploits exchange-based magnetization transfer (MT) between solute and water protons. CEST effects compete with direct water saturation and conventional MT processes, and generally can only be quantified through an asymmetry analysis of the water saturation spectrum (Z-spectrum) with respect to the water frequency, a process that is exquisitely sensitive to magnetic field inhomogeneities. Here it is shown that direct water saturation imaging allows measurement of the absolute water frequency in each voxel, allowing proper centering ofZ-spectra on a voxel-by-voxel basis independently of spatialB0 field variations. Optimal acquisition parameters for this “water saturation shift referencing” (WASSR) approach were estimated using Monte Carlo simulations and later confirmed experimentally. The optimal ratio of the WASSR sweep width to the linewidth of the direct saturation curve was found to be 3.3–4.0, requiring a sampling of 16–32 points. The frequency error was smaller than 1 Hz at signal-to-noise ratios of 40 or higher. The WASSR method was applied to study glycogen, where the chemical shift difference between the hydroxyl (OH) protons and bulk water protons at 3T is so small (0.75–1.25 ppm) that the CEST spectrum is inconclusive without proper referencing. Magn Reson Med, 2008. © 2009 Wiley-Liss, Inc.