Recently discovered muscle-specific m protein is structurally closely related to the X,K-ATPase -subunits but has a number of unique properties such as predominant localization in intracellular stores and lack of association with known X,K-ATPase -subunits upon heterologous co-expression. In this study primary structure of mouse m was determined and developmental regulation of the gene (ATP1B4) was analyzed. The expression is first detected at day 14 of gestation, is sharply increased at day 16, and reaches its maximum at day 18. After birth, the expression quickly decreases and is hardly detectable in adult mice. A more detailed subcellular localization study was undertaken and its results indicate that m is located not only in sarco-endoplasmic reticulum but is concentrated in nuclear envelopes of both prenatal and postnatal skeletal muscles. Immunohistochemical shows that m is specific to myocytes and in both fetal and newborn skeletal muscles many nuclear envelopes are intensively labeled. Accordingly, m is detected by immunoblotting in purified nuclei and nuclear membranes from newborn skeletal muscles. Upon transfection of human rhabdomyosarcoma cell line RD, GFP-tagged m resides intracellularly with significant enrichment in nuclear envelopes, whereas m with transmembrane domain deleted localizes in both cytoplasm and nucleoplasm. The nuclear m apparently is not in association with Na,K-ATPase since we never detected its -subunit in myonuclear membranes. These results indicate that m has a specialized function in mammalian perinatal myocytes, different from functions of other X,K-ATPase -subunits. The unique temporospatial distribution of m protein expression suggests its important role in development of growing skeletal muscle.