Exercise increases the intracellular T(2) (T(2,i)) of contracting muscles. The mechanism(s) for the T(2,i) increase have not been fully described, and may include increased intracellular free water and acidification. These changes may alter chemical exchange processes between intracellular free water and proteins. In this study, the hypotheses were tested that (a) pH changes T(2,i) by affecting the rate of magnetization transfer (MT) between free intracellular water and intracellular proteins, and (b) the magnitude of the T(2,i) effect depends on acquisition mode (localized or nonlocalized) and echo spacing. Frog gastrocnemius muscles were excised and their intracellular pH was either kept at physiological pH (7.0) or modified to model exercising muscle (pH 6.5). The intracellular transverse relaxation rate (R(2,i) = 1/T(2,i)) always decreased in the acidic muscles, but the changes were greater when measured using more rapid refocusing rates. The MT rate from the macromolecular proton pool to the free water proton pool, its reverse rate, and the spin-lattice relaxation rate of water decreased in acidic muscles. It is concluded that intracellular acidification alters the R(2,i) of muscle water in a refocusing rate-dependent manner, and that the R(2,i) changes are correlated with changes in the MT rate between macromolecules and free intracellular water.