The administration of chemotherapy in clinical situations is limited frequently because of the associated toxicity to normal bone marrow cells, gastrointestinal epithelium, and other host tissues. Although nutritional support has been advocated to reduce chemotherapy-related toxicity in cancer patients, few studies substantiate this clinical impression. The current study was performed to determine the role of nutritional status and enteral nutrient intake as determinants of methotrexate (MTX) toxicity in a well-controlled, tumor-bearing animal model. After subcutaneous mammary tumor (AC-33) inoculation, 56 female Lewis/Wistar rats were assigned randomly to one of the following two nutritional regimens for 14 days: (1) protein-depleted chow (PC) (0.03% protein; 4.27 kcal/g) or (2) standard chow (RC) (22.0% protein; 3.50 kcal/g). After 7 days of dietary control, all animals received one of three weight-adjusted doses of MTX (5, 10, or 20 mg/kg intramuscularly [IM] ) or placebo. All animals received leucovorin rescue (0.6 mg IM) at 6 and 24 hours after MTX injection. Improved nutritional status was associated with a significant reduction in objective measures of MTX-related morbidity and mortality. At low doses of MTX (5 and 10 mg/kg), the mean duration of clinical signs of toxicity (i.e., hair loss, lethargy, and diarrhea) and severity of leukopenia were greater in protein-depleted (PD) animals. With high-dose MTX (20 mg/kg), mortality was increased significantly in PD animals (100%) compared with well-nourished animals (0%). Equivalent tumor response was observed in PD and well-nourished animals. Thus, improved nutritional status by enteral nutrition reduced the morbidity and mortality associated with MTX significantly in this tumor-bearing animal model.