A simple and precise method has been developed for the determination of 25-hydroxyvitamin D in 1 ml of human plasma. The method consists of methanol/chloroform extraction, purification by high pressure liquid chromatography and a competitive protein binding assay using vitamin D deficient rat serum. The ethanol extract from vitamin D deficient chick serum was added to the sample before CPBA to eliminate the non-specific interference in the CPBA system as a vitamin D free serum extract. The assay was sensitive to 0.72 ng/ml of plasma. Satisfactory results were obtained in the dilution and recovery tests. The coefficients of variation were 5.8 approximately 9.1% for the within-assay, and 7.4 approximately 10.3% for the between-assay. Plasma concentrations of 25-hydroxyvitamin D in 46 samples of normal human plasma were 21 +/- 10.5 ng/ml (mean +/- SD), and the seasonal variation was demonstrated. Plasma levels for 25-hydroxyvitamin D were high in patients receiving vitamin D2 and low in patients suffering from liver cirrhosis.