The synthesis and secretion of lipids by mammary epithelial cells is a highly ordered process that involves several distinct steps. Triacylglycerols are synthesized in the endoplasmic reticulum and incorporated into microlipid droplets which coalesce into cytoplasmic lipid droplets. These are vectorially transported to the apical plasma membrane where they are secreted into the milk surrounded by a membrane bilayer. The origin of this membrane as well as the mechanism by which cytoplasmic lipid droplets form and become surrounded by membrane is poorly understood. Proteomic analysis of the protein composition of milk fat globules and cytoplasmic lipid droplet has revealed that the endoplasmic reticulum is not only involved in the synthesis of the lipid but also potentially contributes to the membrane component of milk fat globules. The proteins identified suggest possible mechanisms of multiple steps during this process. Completion of the proteome of milk fat globule membranes and cytoplasmic lipid droplets will provide the necessary reporter molecules to follow and dissect the mechanisms of the sorting and ultimate secretion of cytoplasmic lipid droplets.