The effects of nutritional manipulation and subsequent chemotherapeutic treatment upon growth and metabolism of a transplanted rat rhabdomyosarcoma were investigated by in vivo31P NMR spectroscopy. Nutritional manipulation was accomplished by administration of a protein deprived diet containing no protein and 75.5% glucose. After 5 days the protein deprived rats (PD rats) were nutritionally replenished with a normal protein diet containing 27% protein and 47.3% glucose. Twenty-four hours after nutritional replenishment the PD rats and continuously well-fed controls (NP rats) received methotrexate (MTX, 30 mg/kg, i.p.). 31P NMR spectroscopy of the tumors 24 h after MTX administration showed a decreased ratio of nucleoside triphosphates to inorganic phosphate (referred to as ‘ATP/P1 ratio’) in PD rats in contrast to an unchanged ATP/P1 ratio in the NP controls. At the time of MTX administration the PD rats had a significantly lower tumor pH than the NP group (6.75 ± 0.03 [SEM] vs 6.95 ± 0.04; p < 0.02). Tumor response in the PD group was significantly (p < 0.01) enhanced compared to the NP group. These findings indicate that a period of dietary protein deprivation combined with a high glucose load and followed by nutritional replenishment impairs tumor metabolism. The altered metabolic status is expressed by acidification of the tumor and distinct changes in ATP/P1 ratio and appears to relate to an enhanced susceptibility to MTX chemotherapy.