Vascular smooth muscle cell (VSMC) and leukocyte proliferation are central features of atherosclerosis. We developed a method to measure DNA replication, hence cell division, using ²H₂O to label the deoxyribose moiety of newly synthesized DNA in VSMC and atheroma cells from mouse aorta. Cell turnover/proliferation in aortae from normal and apolipoprotein-E knockout mice (ApoE^-/-) was measured. Mice were injected with ²H₂O to reach 2% body water enrichments, then maintained on 4% ²H₂O in drinking water for weeks to months. DNA from the intimal-medial layer of aorta was extracted and hydrolyzed to deoxyribonucleosides. Purified deoxyadenosine was derivitized to pentane-tetraacetate for analysis of ²H enrichment by gas chromatography-mass spectrometry. VSMC proliferation was measurable but slow in adult mice (0.12 ± 0.08% per day) and higher in young mice (0.25 ± 0.08% per day). VSMC delabeling revealed slow ²H-die away in VSMC DNA, confirming the low turnover rate. Atheroma cell proliferation was elevated in ApoE^-/- mice after 15 weeks on low- or high-fat diets, concurrent with histological appearance of atherosclerosis. Validation of the method for VSMC was confirmed by comparing in vitro rat VSMC proliferation rates using ²H₂O to cell counts and bromodeoxyuridine proliferative indices. In summary, proliferation of VSMC and atheroma cells can be quantified reliably and sensitively without radioactivity, and may be an informative biomarker in vascular hyperplastic diseases, including atherosclerosis.