Caseins, a family of acidic phosphoproteins, interact with colloidal calcium phosphate and organize in a supramolecular structure: the casein micelle. The mesostructure of this supramolecular assembly markedly influences its nutritional and technological functionalities. Despite this, the detailed molecular organization and the cellular mechanisms involved in its biogenesis have only been partially established. Based on physico-chemical and biophysical data, numerous models of the casein micelle have emerged but many questions remain about the specific arrangement of the caseins in the micelle, their interactions with the mineral fraction, the role of each casein in the association process and the potential role of aggregation in their intracellular transport to the apical surface of mammary epithelial cell (MEC). To obtain new insight about the intrinsic organization of the native casein micelles, and the mechanisms participating in their elaboration, we have developed an original strategy that takes advantage of the space-time dimension of the building process to study their construction in the secretory pathway of MEC. Our study shows that the primary interactions between caseins take place in the endoplasmic reticulum (ER). Casein association, notably between κ-casein molecules which contain at least one cysteine residue in all species studied, starts with the formation of disulfide bonds in the ER lumen. The aggregation of αS1-casein (homo and/or hetero-association) also apparently starts in the ER, while ß-casein is at most weakly associated with primary casein aggregates. We have also demonstrated that a substantial proportion of αS1-casein is strongly associated with the ER membranes and with those of more distal compartments of the secretory pathway. Our results indicate an important role of dimerization via disulfide bonds in interaction of this casein with membranes. This recent finding most likely explains our previous observation of an accumulation of caseins in the ER of MEC that do not express αS1-casein, and could lead to a mechanistic explanation for the failure of the export of the other caseins to the Golgi apparatus in this context. Furthermore, we have shown that αS1-casein associates with membrane domains resistant to extraction by detergents, domains analogous to the so-called lipid rafts that are heavily involved in protein sorting and transport in the secretory pathway. These findings demonstrate that i) casein micelle biogenesis begins in the ER, ii) ER-to-Golgi transport is a crucial step in casein secretion, iii) αS1-casein is at the central stage of casein export from the ER and iiii) the membrane-associated form of αS1-casein might play a key role in casein transport and/or casein aggregation within the secretory pathway where it might represent a nucleation anchor for casein micelle formation and/or a binding molecule for the cytosolic secretion machinery.