We have used in vivo 31P-nuclear magnetic resonance spectroscopy to study the changes in high-energy phosphates following hyperthermia. Immediately after heating, there is a fall in adenosine triphosphate and apparent intracellular pH and an increase in inorganic phosphate. Following sublethal heating (40° for 15 min), these changes were partial, and they resolved over the subsequent 45 hr. With tumors given severe hyperthermia (47° for 15 min), there was complete disappearance of adenosine triphosphate, with no recovery by 24 hr posttreatment. Qualitatively similar effects were seen after heating of normal leg muscle. The degree of fall of the adenosine triphosphate/inorganic phosphate concentration ratio was directly proportional to the heat dose and to thermal cell kill. 31P-Nudear magnetic resonance spectroscopy may be useful in thermal dosimetry and treatment evaluation following hyperthermia. © 1984, American Association for Cancer Research. All rights reserved.