OBJECTIVE Variability in 25-hydroxyvitamin D (25(OH)D) change following vitamin D supplementation exists. Vitamin D metabolite measurement might assist in predicting 25(OH)D response and also contribute to defining vitamin D adequacy. This study assessed utility of vitamin D metabolite measurements to predict 25(OH)D response and explored the relationship between parathyroid hormone (PTH) and a "vitamin D composite index" comprised of the sum of serum 25(OH) D, cholecalciferol (vitamin D3) and 24,25 dihydroxyvitamin D (24,25(OH)2D). METHODS Sixty-two postmenopausal women were randomized to daily vitamin D3 1,800 IU or placebo for 4 months. Blood was drawn at baseline and after 1 and 4 months. Serum 25(OH)D, vitamin D3, and 24,25(OH)2D were measured by liquid chromatography tandem mass spectroscopy. Free 25(OH)D and PTH were measured by enzyme-linked immunosorbent assay. Repeated measures analysis of variance and regression analyses were performed. RESULTS Baseline 25(OH)D was positively correlated (P<.05) with vitamin D3, 24,25(OH)2D and free 25(OH) D. Daily vitamin D supplementation increased all metabolites (P<.001). Substantial individual variability in 25(OH) D change at 4 months was observed but was unrelated to baseline vitamin D3, 25(OH)D or 24,25(OH)2D. Only body mass index, body weight, and body fat mass was associated with 25(OH)D change at 4 months. The vitamin D composite score was associated with serum PTH, but this association was similar to that observed with 25(OH) D alone. CONCLUSIONS This study does not support measurement of vitamin D metabolites in a composite index to assist in prediction of 25(OH)D response to supplementation. Overweight individuals have less robust 25(OH) D response to supplementation, but variability precludes prediction of the result following daily supplementation. BACKGROUND BMI = body mass index DXA = dual-energy X-ray absorptiometry LC-MS/MS = liquid chromatography tandem mass spectroscopy 25(OH)D = 25-hydroxyvitamin D 24,25(OH)2D = 24,25 dihydroxyvitamin D PTH = parathyroid hormone vitamin D3 = cholecalciferol.