A precise specific method for measuring 25-hydroxycholecalciferol (25-OH CC) in 2 ml of human serum is described. It includes extraction with acetonitrile, separation with Sep-Pak C-18 cartridges, purification through HPLC and a further quantitation by means of a protein-binding assay. An exhaustive study of this protein-binding method has been performed. The sensitivity of the protein-binding assay is 20 pg/tube and the inter and intra-assay coefficients of variation are 9.6% and 8.7% respectively. The precision of the overall process has been assessed by calculating the inter and intra-assay coefficients of variation, 15.5 and 10.4% respectively. Mean serum value of 25-OH CC in normal subjects (18-50 years old, during spring) is 11.2 +/- 5.5 ng/ml.