Islets of Langerhans isolated from experimental animals, such as mice and rats, have contributed much to our understanding of the mechanisms by which pancreatic β-cells secrete insulin in a regulated manner, and this knowledge is important in identifying potential novel therapies for Type 2 diabetes. However, although many of the signal transduction pathways identified in rodent islets are common to humans, some critical differences have been demonstrated experimentally. It is, therefore, essential that experiments are performed using islets isolated from human pancreas to provide robust data defining whether the key observations made in rodents are also applicable to the human situation. The rate-limiting factor in this area of research is the supply of high-quality human islets isolated from pancreases retrieved from organ donors, and the protocols described in this chapter aim to provide methods of maximising information that can be obtained when human islets are maintained in culture.