The principal aim of these studies was to evaluate the utility of isoflurane and halothane for NMR investigations of tumor physiology. In vivo 31P and 2H NMR were used to examine RIF-1 tumors before, during, and (for 31P) after anesthesia. In tumors, halothane decreases blood flow, [PCR]:[NTP], and pH indicated by the Pi chemical shift (pHnmr), while it increases [Pi]:[NTP]; effects consistent with well-established cardiovascular effects of halothane. Isoflurane does not affect tumor blood flow or [PCr]:[NTP], but increases tumor [Pi]:[NTP] and decreases tumor pHnmr. In vivo 31P NMR measurements of normal mouse liver (upper abdomen) indicate that isoflurane has a similar effect in the liver. Although the mechanism for these effects is unknown, observation of a split Pi peak during isoflurane anesthesia suggests that a pool of Pi in a lower pH environment may become evident under isoflurane anesthesia. Regardless of the cause for increased [Pi]:[NTP] and decreased pHnmr, the utility of isoflurane anesthesia for 31P NMR studies of energy metabolism is limited.