Single fibres were dissected from mouse flexor digitorum brevis muscles and subjected to a protocol of eccentric stretches consisting of ten tetani each with a 40 % stretch. Ten minutes later the fibres showed a reduced force, a shift in the peak of the force-length relation and a steepening of the force-frequency relation. Addition of the fluorescent dye sulforhodamine B to the extracellular space enabled the T-tubular system to be visualized. In unstimulated fibres and fibres subjected to 10 isometric tetani, the T-tubules were clearly delineated. Sulforhodamine B diffused out of the T-tubules with a half-time of 18 +/- 1 s. Following the eccentric protocol, vacuoles connected to the T-tubules were detected in six out of seven fibres. Sulforhodamine B diffused out of the vacuoles of eccentrically damaged fibres extremely slowly with a half-time of 6.3 +/- 2.4 min and diffused out of the T-tubules with a half-time of 39 +/- 4 s. Vacuole production was eliminated by application of 1 mM ouabain to the muscle during the eccentric protocol. On removal of the ouabain, vacuoles appeared over a period of 1 h and were more numerous and more widely distributed than in the absence of ouabain. We propose that T-tubules are liable to rupture during eccentric contraction probably because of the relative movement associated with the inhomogeneity of sarcomere lengths. Such rupture raises intracellular sodium and when the sodium is pumped from the cell by the sodium pump, the volume load of Na(+) and water exceeds the capacity of the T-tubules and causes vacuole production. The damage to the T-tubules may underlie a number of the functional changes that occur in eccentrically damaged muscle fibres.