Transsected rat soleus muscles incubated in oxygenated Krebs solution in vitro at 37 degrees C develop prominent vacuolation in the vicinity of the cut ends of muscle fibers which extends further along the fiber with increasing time of incubation. Electron microscopy shows that the vacuoles represent dilated segments of T-tubules. Their formation is prevented if the muscle is not cut, or in the following situations: omission of Na+ from the medium, incubation at 10 degrees C, or in media containing either 2,4-dinitrophenol or ouabain. Vacuole formation is considerably reduced by substituting Cl- with organic anions or by adding 9-anthracene carboxylic acid to the medium. These results suggest that a massive influx of sodium into the muscle fibers through their cut ends results in maximal stimulation of the Na+-K+-ATPase of the T-tubular membrane leading to increasing Na+ concentration in the lumen of T-tubules followed by influx of Cl- to maintain the electrical gradient. The accumulation of these ions leads to the entry of water from either the extracellular space or the sarcoplasm and consequent dilatation of T-tubules. Rapid incorporation of lipids into T-tubular membrane to increase its surface is presumably necessary for this to occur. Similar T-tubular dilatation appears to occur in vivo in surviving portions of muscle fibers adjacent to segmental necrosis.