A liquid chromatography/thermospray mass spectrometry method has been developed and used to measure the plasma levels and half-life of 25-hydroxyvitamin D3 in adults. The mean plasma levels of 25-hydroxyvitamin D3 averaged 40 ng ml-1 (n = 4) in summer and 30 ng ml-1 (n = 6) in winter. The method was also used to determine the half-life of serum 25-hydroxyvitamin D3 in subjects maintained on either high or low-fiber diets who had been given an intravenous infusion of (6,19,19-2H3)25-hydroxyvitamin D3 sufficient to label 5% of their estimated body pools. The half-life was determined to be 10.4 days (n = 4), which is approximately 50% of the currently accepted value of 19 days, determined using radiolabeled methods. This difference may be due to kinetic isotope effects arising as a result of the tritiated compounds being labeled at sites that undergo Cyt-P450-catalyzed oxidations.