Pancreatic β-cells, responsible for the synthesis and secretion of insulin in response to a glucose challenge, are located in the islets of Langerhans. Islets are comprised of a heterogeneous population of endocrine cells, including insulin-producing β-cells (approx. 65–70%), glucagon-secreting α-cells (20–25%), somatostatin-secreting δ-cells, and polypeptide (PP)-secreting cells. Much of the cellular and biochemical information concerning the mechanisms by which glucose stimulates insulin secretion by pancreatic β-cells has been obtained in studies using islets isolated from rodents (1). Rat islets provide an ideal source of insulin-producing tissue to study pancreatic β-cell function as insulin secretion by rat islets closely parallels insulin secretion by human islets and it is possible to obtain a large number of islets (300–600) from a single rat pancreas. With the widespread development of transgenic and gene knockout models, mouse islets represent an ideal system to study specific changes in gene expression on β-cell function. In this chapter, the methods that we routinely use to isolate islets from rat and mouse pancreata are described.