Though virtually all cells store neutral lipids as cytoplasmic lipid droplets, mammary epithelial cells have developed a specialized function to secrete them as milk fat globules. We used the mammary epithelial cell line HC11 to investigate the first steps of the formation of cytoplasmic lipid droplets and to evaluate the potential connections between the lipid and protein synthetic pathways. Cytoplasmic lipid droplets originate from the endoplasmic reticulum and display specific proteins on their surface such as ADRP (adipose differentiation-related protein), TIP47 (tail-interacting protein of 47 kDa) and Cav-1 (caveolin-1). Here we show that unsaturated fatty acids (oleate and linoleate) induce a pronounced proliferation of cytoplasmic lipid droplets and stimulate the synthesis of ADRP but not that of TIP47 or Cav-1. Unexpectedly, the cellular store of b-casein, accumulated under the lactogenic action of prolactin and glucocorticoids, was decreased following cell treatment with unsaturated fatty acids. In contrast, saturated fatty acids (stearate and palmitate) had no significant effect on either cytoplasmic lipid droplet proliferation or cellular level of b-casein. We show that the effect of unsaturated fatty acids on the level of b-casein is posttranslational, requires protein synthesis to occur and is blocked by lysosome inhibitors. We also observe that proteasome inhibitors potentiate b-casein degradation, indicating that it can destroy some unknown cytosolic protein(s) implicated in the process that negatively controls b-casein levels. Finally, our data suggest that the degradation of b-casein occurs via the autophagic pathway.