Down-regulation of the proteasome activator PA28 results in abnormal proteasome activation and has been implicated in the development of intimal hyperplasia (IH) in animal models. Demonstration of proteasome and PA28 expression has not yet been documented in the human vascular system. This study sought to define the distribution of the 20S proteasome and its activator PA28 in human vessels and determine the relationship between the expression of the proteasome and PA28 and the development of atherosclerosis and IH. Vascular biopsies were obtained from 70 patients at the time of surgery, were snap frozen and sectioned in 5-μm sections, and prepared using standard histological techniques. The immunoperoxidase technique was used to identify 20S proteasome and PA28 expression in diseased and normal human arteries and veins as well as in patent bypass grafts with and without IH. Expression was graded by a blinded pathologist (scale: 1-4). Repeat quantification of the immunopositive cells was also performed. Expression of 20S proteasome and PA28 was identified in all vascular tissues examined. The proteins were identified predominately within the cytoplasm of vascular smooth muscle cells and endothelial cells. PA28 was more intensely expressed in quiescent regions of the vessel wall as compared to areas undergoing active proliferation and remodeling. PA28-mediated activation of the proteasome may be necessary to maintain normal cellular homeostasis and prevent excessive cellular proliferation in the human vascular system. Abnormalities of proteasome activation may have a significant role in the development of atherosclerosis and IH.