31P-magnetic resonance spectroscopy (MRS) and a fiberoptic pH meter were used simultaneously to follow the changes in intra- (pHi) and extracellular pH (pHe), respectively, of murine RIF-1 tumors with hyperthermia. Hyperthermia was induced at 34 MHz using the same coil used for MR. The study was carried out until 3.5 hr after hyperthermia. In untreated tumors (n = 29), pHi was always higher than pHe. pHi was reduced after hyperthermia (30 min) at both 42 degrees C and 45 degrees C. pHe registered an increase after 42 degrees C and a decrease after 45 degrees C. The reduction in pHi was larger than the initial differential between pHi and pHe, and the change in pHe was relatively small. Hyperthermia changed the acidity of the intra- and extracellular compartments, such that pHe became more alkaline than pHi by 0.15 +/- 0.13 units after 42 degrees C [pHe (7.20 +/- 0.12) and pHi (7.03 +/- 0.05)], and by 0.12 +/- 0.14 units after 45 degrees C [pHe (6.84 +/- 0.24) and pHi (6.72 +/- 0.19)]. Simultaneous measurements of pH from the intra- and extracellular compartments demonstrated reversal in the pH gradient after hyperthermic treatment.