This study quantifies the enhancement of the therapeutic efficacy of hyperthermia resulting from an acutely acidified and accurately monitored intracellular pH (pHi) in a mouse tumor model in vivo. Metabolic manipulation of the physiology of RIF-1 tumor (subcutaneous, on the hind flanks of female C3H/HeJ mice) achieved by i.p. bolus injection of glucose (glycolytic tumor acidification) or 3-O-methylglucose (non-glycolytic tumor acidification) was monitored by 31P magnetic resonance (31P MR) prior to, during and up to 1 h after localized hyperthermia. The pre-hyperthermia 31P MR-observable metabolic parameter that correlates most strongly with thermal sensitivity is pHi. Thermal sensitivity increases linearly with decreasing pH, regardless of the mechanism (glycolytic or non-glycolytic) of metabolic manipulation. The quantitative relationship is described by log10(SF)/EQ43 = 0.0079 pHi,preHT -0.0606 (R = 0.63, P < 0.0001), where EQ43 is the thermal heat dose delivered to the tumor (in units of equivalent minutes at 42.5°C), pHi,preHT is the intracellular pH immediately prior to hyperthermia, and SF is the surviving fraction. The therapeutic enhancement is not as dramatic as expected based upon previously reported in vitro studies but is generally consistent with other in vivo studies. The method still represents a viable strategy for enhancing the therapeutic efficacy of hyperthermia, especially when used in combination with other therapeutic modalities.