The effects of pentobarbital anesthesia on the energy metabolism of FSaII and MCaIV foot tumors in mice were studied by 31P MRS. Using an 8.5 T spectrometer, in vivo spectra were obtained in 15 animals before and after pentobarbital anesthesia (0.05 mg/g ip). The average phosphocreatine/inorganic phosphate ratios (PCr/Pi) with and without pentobarbital were similar for both tumor histologies. Effects on individual tumors, however, were greater than 20% in 9/15 animals and greater than 50% in 6/15 animals. Pentobarbital anesthesia increased the variability of tumor intracellular pH, and the phosphomonoester/nucleotide triphosphate (PME/NTP) and nucleotide triphosphate/inorganic phosphate ratios (NTP/Pi). When examining the average in a cohort, pentobarbital anesthesia had no significant effect on the PCr/Pi, PME/NTP, NTP/Pi ratios or the pH. However, approximately equal to 50% of individual tumors do have significant changes in these parameters. The anesthesia-induced variability of tumor energy metabolism may explain the decrease in TCD50 observed in previous studies using multifraction radiation.