Calsequestrin (CS) is the low affinity, high capacity calcium binding protein segregated to the lumen of terminal cisternae (TC) of sarcoplasmic reticulum (SR). The physiological role of CS in controlling calcium release from SR depends upon both its intrinsic properties and localization. The mechanisms of CS targeting were investigated in skeletal muscle fibers and C₂C₁₂ myotubes, a model of SR differentiation, using four deletion mutants of epitope (HA)-tagged CS: CS-HA24_NH2, CS-HA2D, CS-HA3D, CS-HAHT, a double mutant of the NH₂-terminus and domain III. As judged by immunofluorescence of transfected skeletal muscle fibers, only the double CS-HA mutant showed a homogeneous distribution at the sarcomeric I band, i.e., it did not segregate to TC; as shown by subfractionation of microsomes derived from transfected skeletal muscles, CS-HAHT was largely associated to longitudinal SR whereas CS-HA was concentrated to TC. In C₂C₁₂ myotubes, as judged by immunofluorescence, not only CS-HAHT but also CS-HA3D and CS-HA2D were not sorted to developing SR. The condensation competence, a property referable to CS oligomerization, was monitored for the several CS-HA mutants in C₂C₁₂ myoblasts, and only CS-HA3D was found able to condensate. Taken together, the results indicate that: i) there are at least two targeting sequences at the NH₂-terminus and domain III of CS, ii) SR-specific target and structural information is endowed in these sequences, iii) heterologous interactions with jSR proteins are relevant for segregation, iv) homologous CS-CS interactions are involved in the overall targeting process, v) different targeting mechanisms prevail depending upon the stage of SR differentiation.