In vivo 31P-nuclear magnetic resonance (NMR) spectroscopy and 133Xe clearance were used to monitor serially ATP content and blood flow, respectively, in C3H/HeJ mouse subcutaneous RIF-1 tumors treated with hyperthermia. There was a prompt decrease in ATP [measured spectroscopically and expressed as the ratio of ATP to Pi (ATP/Pi)]. The slope and magnitude of the change in ATP closely paralleled those of tumor blood flow. Close correlation between these two variables was seen when data were analyzed both by treatment group and by individual mouse. Ligated tumors showed qualitatively and quantitatively similar changes in ATP/Pi and blood flow. RIF-1 cells heated in vitro to a similar degree showed no decrease in ATP. The loss of ATP in subcutaneous RIF-1 tumors heated in vivo was primarily due to disruption of tumor blood flow. These data emphasize the importance of vascular factors to in vivo thermal effects. In vivo 31P-NMR spectroscopy can be used to monitor indirectly vascular effects from hyperthermia. © 1985 Oxfor University Press. All Rights Reserved.