Oxygen and carbon dioxide are known to be heterogeneously distributed in tissues. Extracellular skeletal muscle tissue pH (pHt) also exhibits a spatial variability in vitro, but this has not been examined in vivo. pHt distributions in resting skeletal muscle and the effect of the dispersion of pHt on ischemia and normoxic hypercarbia was therefore studied in an animal model with a multichannel pH microelectrode. Under resting conditions and spontaneous breathing, local pHt (from all animals, n = 10) was found to vary between 6.96 and 7.68 (range), and 70% of the values were within a pH of 7.00-7.32. In each animal the maximum pHt differences (maximum range between the 6 channels of the microelectrode) found were 0.32 ± 0.11 pH units (mean ± SD). During tissue acidosis, induced by ischemia, no significant change in the local pHt differences in each animal was seen. During normoxic hypercarbia a 2-fold increase in pHt variability within each animal was noticed (p < 0.01), which suggests that carbon dioxide and buffering effects of the blood are significant factors for the pHt distribution. The pHt distribution range found is of similar magnitude as previously described in in vitro studies on skeletal muscle. Locally varying pHt levels may be of importance as they will affect cellular H+ extrusion, membrane potential and volume control of different cell populations differentl