Calsequestrin (CS) is the junctional sarcoplasmic reticulum (jSR) Ca2+ binding protein responsible for intraluminal Ca2+ storage. The targeting mechanisms of CS to the jSR are yet to be unraveled. The nine-amino acid epitope of the influenza virus hemoagglutinin (referred to as HA1) was added at the COOH-terminal of CS by polymerase chain reaction cloning. The HA1-tagged CS cDNA was transiently transfected in either HeLa cells, myogenic cell lines, such as C2 and L8 cells, myoblasts of rat skeletal muscle primary cultures, or regenerating soleus muscle fibers of adult rats. The expression and intracellular localization of chimeric CS-HA1 were monitored by epifluorescence and confocal microscopy using either anti-CS antibodies or anti-HA1 antibodies. About 30% of transfected HeLa cells and 20-40% of myogenic cells expressed CS-HA1 into intracellular compartments, such as the perinuclear cisternae of endoplasmic reticulum (ER). Myoblasts of newborn rat skeletal muscles were first transfected and subsequently stimulated to differentiate into myotubes. CS-HA1 was detected in approximately 20% of transfected myotubes and did not affect CS distribution in myotubes. In the soleus muscle of adult rat, intramuscular injection of bupivacaine induced necrosis followed by regeneration. In vivo transfection of HA1-tagged CS cDNA in regenerating skeletal muscles determined expression in a few skeletal muscle fibers; CS-HA1 was localized only in jSR, as judged by confocal microscopy of longitudinal sections. The present results show that chimeric CS-HA1 is correctly sorted to ER/SR compartments and that the free COOH-terminal is not requested for sorting, retention, and segregation of CS to the SR.