We report the identification using monoclonal antibody and the primary structure by cDNA cloning of mitsugumin23, a novel transmembrane protein with a molecular mass of approximately 23 kDa from skeletal muscle sarcoplasmic reticulum. Mitsugumin23 possesses three putative transmembrane segments, and its carboxy-terminal hydrophilic region exhibits sequence similarity with the tail-end portion of the myosin heavy chain. Immunochemical analysis showed that this protein is distributed throughout the outer nuclear membrane and the sarcoplasmic reticulum including the terminal cisternae at the triad junction in skeletal muscle cells. Furthermore, RNA blotting and immunohistochemical experiments demonstrated that mitsugumin23 is distributed among a wide variety of cell types in various tissues. The distribution and primary structure indicate the possibility that mitsugumin23 interacts with cytoplasmic protein(s) and participates in a housekeeping function on the intracellular organelle membranes.