Transverse tubules (TT) are plasma membrane invaginations that propagate action potentials to the inside of the skeletal muscle fiber (Melzer et al 1995). The lipid composition of isolated TT, with 35% cholesterol and 15% sphingomyelin (Anderson, 1998), is equivalent to that of caveolae and lipid rafts (Parton et al. 1997). There are conflicting reports regarding the presence of caveolin-3 in adult skeletal muscle. Some reports indicate that caveolin-3 is present in TT in developing skeletal muscle but not in adult muscle (cf. Ralston & Ploug, 1999). Other groups, however, have reported expression of caveolin-3 in TT from adult skeletal muscle using microscopy (Munoz et al. 1996) or subcellular fractionation techniques (Parton et al. 1997). Here, we investigated the role of caveolin-3 in adult rabbit skeletal muscle fibers. New Zealand male rabbits were sedated with ketamine (50 mg/kg i.m.) and killed by cervical dislocation. By means of subcellular fractionation experiments, we found high levels of caveolin-3 in isolated TT and triads. Following a classical method to isolate rafts/caveolae (Ralston & Ploug, 1999), we isolated in sucrose density gradients light fractions from TT and triads solubilized with Triton X-100 at 4^oC. Light fractions prepared with this method were markedly enriched in caveolin-3 and cholesterol, two markers of caveolae. These results indicate that caveolin-3 is present in TT and triads isolated from adult skeletal muscle fibers, presumably forming part of cholesterol-rich caveola. Light fractions obtained from triads, however, had lower cholesterol contents and appeared at higher sucrose density than light fractions obtained from TT, suggesting that these fractions retain attached sarcoplasmic reticulum (SR) junctional proteins. Additionally, we have analyzed the effect of cholesterol removal on the structure and integrity of isolated triads and TT. Incubation of TT with methyl-β-cyclodextrin released a fraction of the α1-DHPR to the supernatant, suggesting that cholesterol removal disrupts TT integrity.