Adipose tissue has emerged as an important endocrine regulator of glucose metabolism and energy homeostasis. By virtue of the mitochondrial protein uncoupling protein-1 (UCP-1), brown fat additionally plays a unique role in thermoregulation. Interest has focused on this tissue not only as a target for pharmacotherapy of obesity and insulin resistance but also as an endocrine tissue with leptin secretion and high insulin sensitivity. Most studies of adipocytes have been limited either to primary cell culture or to a small number of established cell lines. Recently, we have generated immortalized brown adipocyte cell lines from single newborn mice of different knockout mouse models. These cell lines retain the main characteristics of primary cells including UCP-1 expression. They display sensitive and diverse metabolic responses to insulin and adrenergic stimulation and have proven to be useful in the characterization of UCP regulation and the role of key insulin signaling elements for insulin action. Here, we outline common approaches to the generation of adipose tissue cell lines. Furthermore, we propose that the novel technique of generating brown adipocyte lines from a single newborn mouse will be instrumental in gaining further insight into the role of a broad range of signaling molecules in adipose tissue biology and in the pathogenesis of insulin resistance.