The turnover of adipose tissue components (lipids and cells) and the pathways of adipose lipid deposition have been difficult to measure in humans. We apply here a ²H₂O long-term labeling technique for concurrent measurement of adipose-triglyceride (TG) turnover, cell (DNA) proliferation, and de novo lipogenesis (DNL). Healthy subjects drank ²H₂O (70 ml/day) for 5-9 wk. Subcutaneous adipose tissue aspirates were taken (gluteal, thigh, and flank depots). Deuterium incorporation into TG glycerol (representing all-source TG synthesis), TG palmitate (representing DNL, by mass isotopomer distribution analysis), and DNA (representing cell proliferation) was measured by gas chromatography-mass spectrometry. Subjects tolerated the protocol well, and body ²H₂O enrichments were stable. Mean TG-glycerol fractional synthesis was 0.12 (i.e., 12%) with a range of 0.03-0.32 after 5 wk and 0.20 (range 0.08-0.49) after 9 wk (TG half-life 200-270 days). Label decay measurements 5-8 mo after discontinuing ²H₂O gave similar turnover estimates. Net lipolysis (TG turnover) was 50-60 g/day. DNL contribution to adipose-TG was 0.04 after 9 wk, representing ∼20% of newly deposited TG. Cell proliferation was 0.10-0.17 after 9 wk (half-life 240-425 days). In summary, long-term ²H₂O administration to human subjects allows measurement of the dynamics of adipose tissue components. Turnover of all elements is slow, and DNL contributes ∼20% of new TG.