1. We describe a modified competitive protein-binding assay for 25-hydroxyvitamin D, which does not require the prior separation of vitamin D from 25-hydroxyvitamin D. 2. Bovine albumin was used in the buffer, at a concentration of 1 mg/ml, as a protein stabilizer and lipid solubilizer. Bovine albumin from different manufacturers produced very different non-specific binding of 25-[3H]hydroxycholecalciferol (ranging from 3·3% to 58·9%). 3. The mean concentration of 25-hydroxyvitamin D in sera from normal children in Johannesburg was 76·75 ± 24·75 μnol/ml (30·7 ± 9·9 ng/ml).