Smooth muscle cell (SMC) accumulation in the inner layer of the vessel wall is a key event in the pathogenesis of atherosclerosis in vein grafts, but the origin of the cells in these lesions has yet to be shown. Herein, we use animal models of vein grafts in transgenic mice to clearly identify the sources of SMCs in atherosclerosis. Vena cava segments were isografted to carotid arteries between four types of transgenic mice, including SM-LacZ expressing β-galactosidase (β-gal) in vascular SMCs, SM-LacZ/apoE^−/−, ROSA26 expressing β-gal in all tissues, and wild-type mice. β-gal–positive cells were observed in neointimal and atherosclerotic lesions of all vein segments grafted between LacZ transgenic and wild-type mice. Double staining for β-gal and cell nuclei revealed that about 40% of SMCs originated from hosts and 60% from the donor vessel. This was confirmed by double labeling of the Y-chromosome and α-actin in the lesions of sex-mismatched vein grafts. The possibility that bone marrow cells were the source of SMCs in grafts was eliminated by the absence of β-gal staining in atherosclerotic lesions of chimeric mice. Furthermore, vein SMCs of SM-LacZ mice did not express β-gal in situ, but did so when these cells appeared in atherosclerotic lesions in vivo, suggesting that hemodynamic forces may be crucial for SMC differentiation. Thus, we provide the first evidence of SMC origins in the atherosclerotic lesions of vein grafts, which will be essential for providing insight into new types of therapy for the disease. The full text of this article is available at http://www.circresaha.org.