Measurement of 25-hydroxyvitamin D (25OH-vitD) is used to assess vitamin D status. We developed a high-sensitivity measurement method for 25OH-vitD and assessed the relationship between 25OH-vitD and parathyroid hormone (PTH) in healthy adults. Aliquots (100 microL) of serum were spiked with internal standard, proteins were precipitated, and samples were analyzed by liquid chromatography-tandem mass spectrometry using 2-dimensional chromatographic separation. Total imprecision was less than 10%, and the limit of quantitation was 1.0 ng/mL. We determined the distribution of concentrations of 25OH-vitD(2) and 25OH-vitD(3) in healthy adults using samples collected during winter and summer and evaluated the association between 25OH-vitD and PTH. The difference between median concentrations of 25OH-vitD in samples collected during winter and summer was 11 ng/mL (27 nmol/L). Statistically significant differences in concentrations of PTH were observed between groups of samples with 25OH-vitD less than 11 (27 nmol/L) and 11 to 15 ng/mL (27-37 nmol/L) and between groups with 25 to 30 (62-75 nmol/L) and more than 40 ng/mL (100 nmol/L). Among the advantages of this method are its high sensitivity and specificity.