We conducted two safety studies of 25-hydroxycholecalciferol [25(OH)D3] in poultry broilers at levels ranging from 1 to 200 times those commonly used for cholecalciferol (vitamin D3) supplementation in the industry. In the first experiment, 1-d-old male and female broiler chickens were fed commercial diets containing either vitamin D3 or 25(OH)D3 at concentrations of 69, 207, and 690 micrograms of 25(OH)D3/kg of feed. The second experiment compared effects of 25(OH)D3 and vitamin D3 on performance and survival of broilers at levels ranging from 1 to 200 times the basal level of 69 micrograms/kg feed. When 25(OH)D3 was fed in equal amounts (wt/wt) to vitamin D3, there was an increase in body weight and a decrease (improvement) in adjusted feed efficiency in both experiments, but the changes were significant only in the first experiment. In the first experiment, serum 25(OH)D3 concentrations increased from 13.3 +/- 4.3 to 42.5 +/- 18 ng/mL in birds fed vitamin D3 or 25(OH)D3, respectively, and rose to 246 +/- 38 ng/mL in birds fed the highest level of 25(OH)D3. Tissue 25(OH)D3 concentrations were much lower than serum concentrations and were highly correlated to the latter, regardless of dietary treatment. In Experiment 2, there was some evidence of renal calcification in birds fed 25(OH)D3 at 10 times the basal level, whereas dietary levels of vitamin D3 of 50 times the basal level were required to show some evidence of renal calcification. On the basis of both renal calcification and body weight, the present studies would suggest that 25(OH)D3 is 5 to 10 times more toxic than vitamin D3.