Antitumor effects of hyperthermia are enhanced by lowering the pH in the tumor tissue with administration of glucose. This decreased pH in the tumor tissue with glucose administration was determined using mouse experimental tumors. 31P-MRS microelectrodes were used for the measurement of pH. By using these two measurement methods, time course change in the tumor tissue was determined in the controls and the groups treated with 6 g/kg of intraperitoneal glucose. The determination of pH with 31P-MRS was calculated from the chemical shift of the peak of creatine phosphate (Pcr) and that of inorganic phosphate (Pi). Following glucose administration, the tumor tissue showed a decrease of 0.3 pH units with the microelectrode method, but did not show any significant decrease in pH with the MRS determination. This finding suggested that 31P-MRS showed intracellular pH (pHi) due to the localization of Pi and that the microelectrode indicated interstitial or extracellular pH (pHe). The ATP/Pi ratio obtained in tumor tissue 24 h after heat treatment (with, without glucose) was correlated with tumor inhibition.