The transverse tubules (T-tubules) of mammalian cardiac and skeletal muscles are invaginations of the sarcolemma. They play a crucial role in excitation-contraction coupling as well as in intracellular signaling and in regulation of glucose transport. The biochemical purification of T-tubule membranes is a difficult task, and membrane fractions enriched in transverse tubules are usually contaminated with other cell-surface and intracellular membranes. This unit includes methods that permit the isolation and purification of T-tubules from skeletal muscle.