Recent studies indicate that junctophilin (JP) subtypes contribute to the formation of the junctional membrane complexes between the plasma membrane and the endoplasmic/sarcoplasmic reticulum (ER/SR) in excitable cells. Cardiac muscle contains the diad, in which the transverse (T) tubule of the invaginated cell membrane is closely associated with the SR membrane, and skeletal muscle bears the triad, in which the T-tubule is associated with two SR membranes on the both sides. Among defined JP subtypes, JP-2 is specifically expressed in cardiac muscle, while skeletal muscle cells contain both JP-1 and JP-2. These observations, together with other findings, suggest that the triad might be constructed in a JP-1-dependent manner after the achievement of JP-2-mediated diad formation during skeletal muscle maturation. In this study using transgenic mice, we examined whether the triad can be formed when JP-1 is additionally expressed in cardiac muscle. Immunochemical analysis demonstrated co-expression of JP-1 and JP-2 in cardiac myocytes from the transgenic mice. In cardiac muscle expressing JP-1, abnormal junctional membranes were frequently observed under the electron microscope, in which the T-tubules were rolled up with the SR membranes at several turns, but authentic triad formation could not be detected. Therefore, ectopic JP-1 expression cannot convert the diad to the triad in cardiac myocytes. The present results suggest that triad formation requires an as yet unknown skeletal muscle-specific mechanism, in addition to the JP subtypes.