The majority of investigations of the transverse tubules (T-system) of skeletal muscle have been devoted to their role in excitation-contraction coupling, with particular reference to contact with the sarcoplasmic reticulum and the mechanism of Ca2- release. By contrast, this review is concerned with structural and functional aspects of the vacuolation of T-tubules. It covers experimental procedures used in reversible vacuolation induced by the efflux-influx of glycerol and other small nonelectrolytes, sugars, and ions. The characteristics of the phenomenon, associated alterations in muscle function, and the swelling of analogous structures in nonmuscle cells are considered. Possible functions of reversible vacuolation in water balance, transport, membrane repair, muscle pathology, and fatigue are considered, and the potential application of reversible vacuolation in the transfection of skeletal muscle is discussed. In relation to the possible mechanisms involved in reversible vacuolation, particular attention is given to the dynamic and structural aspects of the opening and closing of T-tubules, the origin of vacuolar membranes, and the localized character of tubular swelling.