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Although subtle magnetic susceptibility variations caused by iron and myelin seem to be important to this contrast, recent research on protein solutions suggests that chemical exchange between water and macromolecular protons may contribute substantially to the observed gray\u2010white matter frequency contrast. To investigate this, we performed spectroscopic MRI experiments at 14 T on samples of fixed human visual cortex and fresh pig brain. To allow direct observation of any exchange\u2010induced frequency shifts, these samples were soaked in reference chemicals (TSP and dioxane) that are assumed not to be involved in exchange. For both fresh and fixed tissues and with both reference chemicals, substantial negative exchange\u2010induced gray\u2010white matter frequency contrast (\u20136.3 to \u201313.5 ppb) was found, whereas intracortical contrast was negligible. The sign of the gray\u2010white matter exchange\u2010induced frequency difference was opposite to the overall gray\u2010white matter frequency difference observed in vivo. This suggests that exchange contributes to, but is not sufficient to explain, the frequency contrast in vivo and tissue susceptibility differences may have a greater contribution than previously thought. The exchange\u2010dependent contribution may report on tissue chemical composition and pH. 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