1. A competitive protein-binding assay for plasma 25-hydroxycholecalciferol was developed, using Sephadex LH-20 columns for the isolation of the sterol and a partially purified protein from rat serum as the binding protein.

2. The mean plasma 25-hydroxycholecalciferol in eighteen male and female normal volunteers was 38 ± SD 14 pmol/ml (15–2 ± 5–6 ng/ml).

3. Levels of 25-hydroxycholecalciferol in plasma of four male volunteers maintained for 2 months on a diet supplemented with 26·1 nmol (10 μg) of cholecalciferol a day were found to be significantly higher.

4. High correlation was established between plasma 25-hydroxycholecalciferol values obtained by the competitive protein-binding assay and values obtained by bioassay.

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