Competition by 24,25-dihydroxycholecalciferol in the competitive protein binding radioassay of 25-hydroxycalciferol

24,25-dihydroxycholecalciferol (24,25-(OH)2D3) is equipotent to 25-hydroxycholecalciferol in the displacement of 3H25-OHD3 from rat serum binding sites, and is extracted by the ethanol procedure recommended in non-chromatographic competitive protein binding radioassays for 25-OHD. Serum 24,25-(OH)2D content was measured following 24,25-(OH)2D3 isolation from lipid extracts by liquid-gel partition chromatography. Since normal serum 24,25-(OH)2D concentration is appreciable, non-chromatographic 25-OHD assays most probably overestimate serum 25-OHD levels as a result of their recognition of 24,25-(OH)2D3.