Pathogenesis of in-stent restenosis remains poorly understood because information from human histopathologic studies is scarce. We used an improved saw-grinding and cutting method on methacrylate-embedded samples containing metal stents, which allows in situ hybridization and immunohistochemical analysis of in-stent restenosis. Twenty-one samples were collected 3 hours to 3 years after stenting from 6 patients aged 36 to 81 years. Except in very early samples collected within hours after the stent deployment, neovascularization was present in all segments studied. At advanced stages, extensive neovascularization was located mainly at the luminal side of the stent struts and was only rarely accompanied by inflammatory cells. The neovessels colocalized with vascular endothelial growth factor (VEGF)-A mRNA and protein expression as well as with iron deposits and oxidation-specific epitopes, which imply the presence of chronic oxidative stress. VEGF-A expression was detected in the same areas containing macrophages, endothelial cells, and, to a lesser extent, smooth muscle cells, which also showed platelet-derived growth factor-BB expression. We conclude that in-stent restenosis features neovascularization, VEGF-A and platelet-derived growth factor-BB expression, and iron deposition, which is most probably derived from microhemorrhages. These mechanisms may play an important role in the development of neointimal thickening and could provide useful targets for the prevention and treatment of in-stent restenosis.