The primary amino acid sequence for a highly abundant junctional sarcoplasmic reticulum glycoprotein (triadin) has been deduced from the cDNA sequence. Based on both biochemical analysis and the predicted amino acid sequence we suggest that this protein is an intrinsic membrane glycoprotein containing a single transmembrane domain that separates the protein into cytoplasmic and luminal domains. The cytoplasmic domain is proposed to contain the amino-terminal 47 amino acids. The remainder of the protein including the carboxyl terminus is proposed to be found within the lumen of the sarcoplasmic reticulum and contains an extremely high concentration of basic residues. Protease analysis of intact triads was consistent with the topological predictions. Western and Northern blots suggest that the protein is specifically expressed in skeletal muscle and not cardiac muscle or brain. The abundance and localization of this protein suggest that it plays an important regulatory or structural role in excitation-contraction coupling in skeletal muscle.