Cholecalciferol (vitamin D) deficiency rickets remains an occasional problem in poultry. Diagnosis currently relies on analysis of feed and histopathological examination of bone. These experiments were designed to provide data that might allow diagnosis of cholecalciferol deficiency on the basis of plasma concentrations of 25-hydroxycholecalciferol, a circulating metabolite of cholecalciferol. Day-old broiler chicks were fed corn-soybean meal or purified ingredient cholecalciferol-deficient diets supplemented with 0, 5, 10, 15, 20, 25, 37.5 or 75 micrograms cholecalciferol/kg diet. Plasma and bone samples were collected 21 d later. Chicks fed the unsupplemented purified ingredient diet became truly deficient, having no detectable plasma concentrations of the cholecalciferol metabolites 25-hydroxycholecalciferol, 1,25-dihydroxycholecalciferol, or 24,25-dihydroxycholecalciferol. Chicks fed the corn-soybean meal diet without supplementation had low but detectable concentrations of both 25-hydroxycholecalciferol and 1,25-dihydroxycholecalciferol in plasma. Body weight, bone calcium and bone phosphorus concentrations of chicks fed the corn-soybean meal diet suggest that the cholecalciferol requirement of broiler chicks is at least 10 micrograms/kg diet. At this dietary level of cholecalciferol, plasma 25-dihydroxycholecalciferol concentration was 12.5 nmol/L. One hundred percent of the theoretical maximal response in body weight and bone calcium content was seen at 20 micrograms cholecalciferol/kg diet, which increased plasma 25-hydroxycholecalciferol concentration to 25 nmol/L in the chicks fed the corn-soybean meal diet. These data provide a nomogram of plasma 25-dihydroxycholecalciferol concentration that can be expected from including different concentrations of cholecalciferol in the diet, and also offer a means of diagnosing cholecalciferol deficiency in field cases of tickets.