We describe a simultaneous assay for the principal vitamin D metabolites: 25-hydroxyvitamin D, 24-25-dihydroxyvitamin D, and 1,25-dihydroxyvitamin D. Special attention has been paid to simplification of the extensive extraction and purification procedures used in previously described simultaneous assays. All three metabolites were isolated with a single extraction step, followed by only one gradient liquid-chromatographic procedure. For final quantitation we used competitive protein binding assays, involving readily available binding proteins and commercially purchased tritiated vitamin D metabolites. Concentrations in the plasma of healthy subjects (mean age, 27 years), sampled during December were 51 (SD 17) nmol/L, 4.1 (SD 1.3) nmol/L, and 124 (SD 26) pmol/L for 25-hydroxyvitamin D, 24,25-dihydroxyvitamin D and 1,25-dihydroxyvitamin D, respectively. Intra- and interassay CVs for the three metabolites were 4.4 and 3.9%, 6.7 and 8.0%, and 7.0 and 4.8%, respectively.