Composition of diet may influence growth, diseases, tumor rates, and responses to chemical treatment. Since 1980 the NIH-07 open formula nonpurified diet has been the selected diet for the National Toxicology Program (NTP) toxicity and carcinogenicity studies in rodents. Studies with nonpurified experimental diets with lower protein and higher fat and fiber than the NIH-07 diet indicated that the diet for Fischer-344 (F344) rats in long-term studies could be modified to decrease the severity of chronic diseases and to decrease/delay the development of spontaneous tumors. Based on the results of these studies a new open formula nonpurified diet designated as NTP-2000 was formulated to contain approximately 14.5% protein, approximately 8.5% fat, and approximately 9.5% fiber. Corn, wheat, and wheat middlings contribute to about 60% of the ingredients; soybean meal, fish meal, and alfalfa meal are the additional sources of protein; purified cellulose, oat hulls, and alfalfa meal are the major sources of fiber; and soy oil and corn oil are the major sources of fat in the NTP-2000 diet. The Ca:P ratio and mineral and vitamin concentrations were reformulated based on AIN-93 and NRC-95 recommendations. The NIH-07 and the NTP-2000 diets were fed to groups of 6-week-old F344 rats for 13 weeks and evaluated for growth patterns, food and water consumptions, hematology and clinical chemistry parameters, and organ weights and pathological changes. Growth patterns and body weights were similar for both diets. Food consumptions were slightly higher and water consumptions were slightly lower for the groups fed NTP-2000 diet. There were no differences in hematological parameters between the groups fed the above diets. Serum levels of cholesterol, alkaline phosphatase, and 5' nucleotidase were slightly higher in groups fed the NTP-2000 diet possibly due to higher fat content of this diet. However, the serum triglyceride levels were slightly lower in groups fed the NTP-2000 diet and it may be related to higher fiber content of the NTP-2000 diet. The liver and kidney weights of the groups fed NTP-2000 diet were significantly lower possibly due to lower protein content of this diet and lower protein consumption associated changes in Phase I and Phase II drug metabolizing enzyme systems. The adrenal weights were also lower in groups fed the new diet. The NTP-2000 diet prevented nephrocalcinosis and decreased the severity of nephropathy and cardiomyopathy, the common lesions of F344 rats in 13-week studies. These results indicate that the NTP-2000 diet is adequate for growth and maintenance of rats and appears to prevent or decrease the severity of diet-associated lesions.