This report investigated in vivo turnover kinetics of marrow hematopoietic progenitors and precursors using a recently developed stable isotope–mass spectrometric technique (SIMST). Human subjects were administered a 2-day infusion of 6,6-[²H₂]-glucose, a nontoxic stable isotope-labeled form of glucose, which becomes incorporated into DNA of all S-phase cells. The percent [²H₂]-glucose incorporated into DNA in the form of [²H₂]-deoxyadenosine (%[²H₂]-dA enrichment) was determined by gas chromatography–mass spectrometry. The rate constant of replacement of unlabeled by labeled DNA strands (labeling kinetics) was used to calculate population turnover kinetics of CD34⁺ cells, CD133⁺ cells, and CD133^–CD34⁺ cells. The observed mean replacement half-life (t_1/2) was 2.6 days for CD34⁺ cells, 2.5 days for CD133^–CD34⁺ cells, and 6.2 days for CD133⁺ cells. Results from the estimated rate constant of replacement of labeled by unlabeled DNA (delabeling kinetics) also demonstrated slower turnover rates for CD133⁺ cells than for CD133^–CD34⁺ cells. Although there was a relatively rapid initial decrease in the %[²H₂]-dA enrichment, low levels of labeled DNA persisted in CD34⁺ cells for at least 4 weeks. The results indicate the presence of subpopulations of CD34⁺ cells with relatively rapid turnover rates and subpopulations with a slower t_1/2 of 28 days. Results also demonstrate that in vivo [²H₂]-glucose-SIMST is sensitive enough to detect differences in turnover kinetics between erythroid and megakaryocyte lineage cells. These studies are the first to demonstrate the use of in vivo [²H₂]-glucose-SIMST to measure in vivo turnover kinetics of subpopulations of CD34⁺ cells and precursors in healthy human subjects.