Exercise-induced decreases in the 1 H transverse relaxation rate (R 2 ) of muscle have been well documented, but the mechanism remains unclear. In this study, the hypothesis was tested that R 2 decreases could be explained by pH decreases and apparent intracellular volume (V i ) increases. 31 P and 1 H spectroscopy, biexponential R 2 analysis, and imaging were performed prior to and following fatiguing exercise in iodoacetate-treated (IAA, to inhibit glycolysis), NaCN-treated (to inhibit oxidative phosphorylation), and untreated frog gastrocnemii. In all exercised muscles, the apparent intracellular R 2 (R 2i ) and pH decreased, while intracellular osmolytes and V i increased. These effects were larger in NaCN-treated and untreated muscles than in IAAtreated muscles. Multiple regression analysis showed that pH and V i changes explain 70% of the R 2i variance. Separate experiments in unexercised muscles demonstrated causal relationships between pH and R 2i and between V i and R 2i . These data indicate that the R 2 change of exercise is primarily an intracellular phenomenon caused by the accumulation of the end-products of anaerobic metabolism. In the NaCN-treated and untreated muscles, the R 2i change increased as field strength increased, suggesting a role for pH-modulated chemical exchange. Key words: skeletal muscle; exercise; hydrogen-ion concentration; osmolarity; nuclear magnetic resonance Exercise-induced increases in the apparent 1 H transverse relaxation time (T 2 ) of muscle water have been well documented. The amount of T 2 change increases as exercise intensity increases; T 2 plateaus 3-4 min after the onset of exercise (1). Following isometric leg extension to fatigue, T 2 recovery follows an approximately exponential time course and is complete after ϳ35 min. (2). The increase in signal intensity from active muscles in T 2 -weighted images allows muscle activity to be detected reliably and noninvasively, which may aid in the placement of regions of interest or surface coils in spectroscopic studies (2,3). With a full understanding of the mechanism(s) of the T 2 change, this phenomenon may also be useful in functional studies of muscle activation during exercise (e.g., Refs. 4 -6), noninvasive studies of pathologic conditions in which the T 2 response to exercise is altered (e.g., Refs. 7 and 8), and as a means of studying noninvasively those physiological changes that increase T 2 .The most universally offered and best-studied explanation for the T 2 increase during exercise is the concomitant increase in muscle volume. However, the mechanism of the T 2 increase is more complex than total water accumulation. For example, recovery of the muscle's anatomical cross-sectional area is faster than T 2 recovery (2), and enhancement of the extracellular fluid volume increase during exercise is not proportional to the T 2 increase (9). A further complication is that both ex vivo amphibian (10) and in vivo mammalian (11) studies suggest that exchange between the intra-and extracellular spaces in muscle is sl...