A method is presented for measurement of triglyceride (TG) synthesis that can be applied to slow-turnover lipids. The glycerol moiety of TG is labeled from ²H₂O, and mass isotopomer distribution analysis (MIDA) is applied. Mice and rats were given 4-8% ²H₂O in drinking water; TG-glycerol was isolated from adipose and liver during ≤12-wk of ²H₂O labeling. Mass isotopomer abundances in the glycerol moiety of TG were measured by GC-MS. The combinatorial pattern of isotopomers revealed the number of H atoms in glycerol incorporating label from ²H₂O (n) to be 3.8–4.0 of a possible 5 for adipose tissue and 4.6–4.8 for liver TG. Hepatic TG-glycerol in fact reached 97% predicted maximal value of label incorporation (4.4–4.6 × body ²H₂O enrichment), indicating near-complete replacement of the liver TG pool. Label incorporation into adipose tissue revealed turnover of mesenteric TG to be faster (k = 0.21 day^–1) than other depots (k = 0.04–0.06 day^–1) in mice. TG isolated from subcutaneous depots of growing adult rats plateaued at 85–90% of calculated maximal values at 12 wk (k = 0.05 day^–1), excluding significant dilution by unlabeled α-glycerol phosphate. Turnover of plasma TG, modeled from ²H incorporation over 60 min, was 0.06 min^–1 (half-life 11.5 min). In summary, use of ²H₂O labeling with MIDA of TG-glycerol allows measurement of new α-glycerol phosphate-derived TG synthesis and turnover. The hypothesis that mesenteric TG is more lipolytically active than other depots, previously difficult to prove by isotope dilution techniques, was confirmed by this label incorporation approach.