In this protocol, we describe a method for isolation and culture of smooth muscle cells derived from the adult rat (or mouse) superior mesenteric artery. Arterial myocytes are obtained by enzymatic dissociation and established in primary culture. The cultured cells retain expression of smooth muscle-specific α-actin and physiological responses to agonists. Cultured arterial myocytes (prepared from wild-type or transgenic animals) provide a useful model for studying the regulation of a wide range of vascular smooth muscle responses at the cellular and subcellular levels. Plasmids, RNA interference and antisense oligodeoxynucleotides can be readily introduced into the cells to alter protein expression. Fluorescent dyes can also be introduced to visualize a variety of activities, some of which may be specific to vascular smooth muscle cells. This protocol requires about 3 h on each of 2 consecutive days to complete.