Vascular smooth muscle cells (VSMCs) are the cellular components of the normal blood vessel wall that provides structural integrity and regulates the diameter by contracting and relaxing dynamically in response to vasoactive stimuli. The differentiated state of the VSMC is characterized by specific contractile proteins, ion channels, and cell surface receptors that regulate the contractile process and are thus termed contractile cells. In addition to these normal functions, in response to injury or during development, VSMCs are responsible for the synthesis of extracellular matrix proteins, become migratory and proliferate. This phenotype has been termed synthetic cells. To better understand the mechanisms regulating these and other processes, scientists have depended on cultured cells that can be manipulated in vitro. In this chapter, we will discuss in detail the explant method for isolation of VSMC and will compare it to the enzymatic digestion method. We will also briefly describe methods for characterizing the resulting cells.