OBJECTIVE To determine the effects of the nitric oxide synthase inhibitor, nitro-L-arginine on energy metabolism in transplantable and spontaneous murine tumors. METHODS The responses of the transplantable murine tumor SCCVII/Ha and a range of spontaneously arising murine mammary adenocarcinomas to 10 mg/kg IV nitro-L-arginine were examined using in vivo 31P magnetic resonance spectroscopy (MRS). The influence of Hypnorm/Hypnovel anesthesia on the response to nitro-L-arginine was also determined in the SCCVII/Ha tumors. Data were expressed as changes in the inorganic phosphate peak area relative to the sum of all peak areas from the 31P MR spectrum, or Pi/total. RESULTS Nitro-L-arginine at 10 mg/kg IV increased Pi/total 2-3-fold in the SCCVII/Ha tumors for at least 2 h after administration, in both anesthetized and nonanesthetized mice, consistent with increased tumor hypoxia. Similar increases in Pi/total were observed after 10 mg/kg IV nitro-L-arginine in 13 spontaneous murine tumors from three different mouse strains, where anesthetic was used. CONCLUSIONS The results indicate that tumor metabolism may be modified by an inhibitor of nitric oxide synthesis, that this modification occurs in both transplantable and spontaneous murine tumors and is not affected by anesthetic.