Serum concentrations of 25-hydroxyvitamin D (25-OH-D) and 24,25-dihydroxyvitamin D [24,25-(OH)2-D] were measured in lipid extracts of 1 ml of human sera by a competitive protein binding assay. 25-OH-D and 24,25-(OH)2-D were isolated from the lipid extracts by Sephadex LH-20 column chromatography. The 24,25-(OH)2-D fraction on the Sephadex LH-20 columns were further purified by high pressure liquid chromatography prior to assay. Serum levels of 25-OH-D and 24,25-(OH)2-D in normal males were similar to those in normal females throughout each age group. Serum levels of 25-OH-D and 24,25-(OH)2-D in newborns were 8.28 .+-. 1.68 ng/ml (mean .+-. SD) and 0.55 .+-. 0.16 ng/ml, respectively, which were significantly (P < 0.001) lower than those in adults (21.3 .+-. 4.8 ng/ml of 25-OH-D and 1.55 .+-. 0.31 ng/ml of 24,25-(OH)2-D). A seasonal variation was demonstrated in serum levels of 24,25-(OH)2-D as well as those of 25-OH-D. Serum levels of 24,25-(OH)2-D were highly correlated (correlation coefficient = 0.884, P < 0.001) with those of 25-OH-D in normal human subjects, and the percentage ratio of 24,25-(OH)2-D/25-OH-D was 7.4 .+-. 1.4% (mean .+-. SD), irrespective of age or seasonal variations.