1. In this study a new method has been used to measure interstitial pH continuously in human muscle during graded exercise. Human subjects performed 5 min of one-legged knee-extensor exercise at power outputs of 30, 50 and 70 W. Muscle interstitial pH was measured continuously in microdialysis dialysate using the pH-sensitive fluorescent dye 2',7'-bis-(2-carboxyethyl)-5-(and -6)-carboxyfluorescein (BCECF). 2. The mean interstitial pH at rest was 7.38 +/- 0.02. Interstitial pH gradually reduced during exercise in a nearly linear manner. The mean value (range) of the lowest interstitial pH at 30, 50 and 70 W exercise was 7.27 (7.18-7.34), 7.16 (7.05-7.24) and 7.04 (6.93-7.12), respectively. 3. The lowest pH was obtained 1 min after exercise, irrespectively of the workload, after which interstitial pH recovered in a nearly exponential manner. The mean half-time for recovery was 5.2 min (range 4.1-6.1 min). The changes in interstitial pH exceeded the changes in venous blood pH. 4. The present study showed that interstitial pH decreased during exercise in relation to intensity. These pH changes could have implications for blood flow regulation as well as for modulations of membrane transport systems.