Caveolin-3, the muscle-specific isoform of the caveolae-associated protein caveolin, is often thought to be localized exclusively in the surface membrane in mature fibers and associated with transverse (t)-tubular system only transiently during development. Skeletal muscle fibers present a model where the surface membrane (sarcolemma) can be completely separated from the cell by mechanical dissection. Western blotting of matching portions of individual fibers from adult rat muscle in which the sarcolemma was either removed (skinned segment), or left in place (intact segment), revealed that > or = 70% of caveolin-3 is actually located deeper in the fiber rather than in the sarcolemma itself. Triton solubility of caveolin-3 was no different between sarcolemmal and t-tubule compartments. Confocal immunofluorescence microscopy showed caveolin-3 present throughout the t-system in adult fibers, with 'hot-spots' at the necks of the tubules in the sub-sarcolemmal space. A similar representation was seen for the muscle specific voltage-dependent sodium channel Nav1.4 and it was found that at least some Nav1.4 co-immunoprecipitated with caveolin-3 in skinned muscle fibers. The caveolin-3 hot-spots just inside the opening of t-tubules may form regions that localize ion channels and kinases at the key place needed for efficient electrical transmission into the t-tubules as well as for other signaling processes.