The dihydropyridine receptors (DHPR) and the ryanodine receptors (RyR) are well-characterized proteins of the triad junctions of skeletal muscle fibers. Recently, a newly discovered 95-kDa protein, triadin, has been purified from rabbit skeletal muscle heavy sarcoplasmic reticulum (SR) vesicles. WE have used indirect immunogold EM to localize triadin to the junctional face of the SR in isolated triads. In addition, we have used indirect immunofluorescence to localize triadin in relation to the DHPR and the RyR in adult and developing rat skeletal muscle. In double immunolabelling experiments of longitudinally oriented adult rat skeletal muscle tissue, triadin-specific and RyR-specific antibodies resulted in a characteristic striated staining pattern. The staining arising from these antibodies completely overlapped when examined by computer analysis of digitized laser scanning confocal microscopy images. A similar result was obtained in double staining experiments using antibodies raised against the DHPR and the RyR suggesting that all three proteins are located in the triads in situ. The developmental expression of the three triad proteins was examined using double labeling of skeletal muscle tissue from several fetal and early postnatal ages. The staining patterns of triadin, RyR, and DHPR antibodies were overlapping throughout development, suggesting that from their earliest appearance the three proteins are components of the triads.