Localized pH changes have been suggested to occur in the brain during normal function. However, the existence of such pH changes has also been questioned. Lack of methods for noninvasively measuring pH with high spatial and temporal resolution has limited insight into this issue. Here we report that a magnetic resonance imaging (MRI) strategy, T(1) relaxation in the rotating frame (T(1)ρ), is sufficiently sensitive to detect widespread pH changes in the mouse and human brain evoked by systemically manipulating carbon dioxide or bicarbonate. Moreover, T(1)ρ detected a localized acidosis in the human visual cortex induced by a flashing checkerboard. Lactate measurements and pH-sensitive (31)P spectroscopy at the same site also identified a localized acidosis. Consistent with the established role for pH in blood flow recruitment, T(1)ρ correlated with blood oxygenation level-dependent contrast commonly used in functional MRI. However, T(1)ρ was not directly sensitive to blood oxygen content. These observations indicate that localized pH fluctuations occur in the human brain during normal function. Furthermore, they suggest a unique functional imaging strategy based on pH that is independent of traditional functional MRI contrast mechanisms.