Ca²⁺ storage and release in muscle cells are controlled by a complex of junctional sarcoplasmic reticulum (jSR) proteins, that includes the calcium-binding protein calsequestrin (CSQ), the Ca²⁺-release channel (ryanodine receptor or RyR) and two transmembrane proteins that bind to RyR: junctin (JNC) and triadin (Tr). The relationship between CSQ and JNC, and their contributions to the architecture of the jSR vesicle was studied in transgenic mice with combined overexpression of CSQ and JNC. We find that CSQ, on its own, has a diffuse disposition in the sarcoplasmic reticulum (SR) lumen. Overexpression of JNC results in a tighter packing of CSQ in proximity of the SR membrane, presumably due to the binding of CSQ to the membrane by JNC. Quantitative and qualitative analysis of structural changes in the overexpressing as well as in the normally differentiating myocardium illustrate the synthetic pathways and the events in the targeting and delivery of CSQ and JNC to the jSR of the differentiating cardiac myocyte. CSQ is delivered from the Golgi to the SR, where it buds out into precursors of the jSR vesicles. JNC reaches the jSR vesicles directly, but its arrival is delayed relative to CSQ.