The Golgi apparatus is a membranous organelle that modifies and packages proteins and lipids into transport carriers and sends them to the proper locations in the cell. The study of Golgi structure and function can be facilitated by the isolation of this organelle from homogenates of tissues or cells. Liver cells have abundant Golgi membranes because they actively secrete proteins and lipids; therefore, liver tissue is often the preferred source. In this protocol, Golgi membranes are purified from rat liver homogenate by two sequential sucrose gradients. The relative yield of the prepared Golgi stacks is then assessed by measuring the increase in activity of a Golgi marker enzyme, β-1,4-galactosyltransferase, over that of the total liver homogenate. A typical preparation can yield Golgi membranes that are purified 80- to 100-fold over the homogenate, and the majority (60%–70%) retain their stacked nature.