Smooth muscle cells (SMCs) are key players in the pathogenesis of atherosclerosis and restenosis; however, they are also important in formation and development of de novo blood vessels during vasculogenesis and angiogenesis. Vascular SMCs can be formed by proliferation of existing SMCs, maturation of pericytes, or putative smooth muscle progenitor cells, thereby contributing to development of atherosclerotic plaques and angiogenic processes. Modulation of SMC phenotype is now recognised as a key event in the development of vascular diseases. This chapter describes the isolation and culture of vascular SMCs and pericytes from human and animal blood vessels for in vitro studies.