The mammary gland from midpregnant rabbits has been dissociated into individual cells by enzymatic digestion, divalent cation chelation, and gentle shearing. A heterogeneous cell population is obtained, comprising approximately 60% parenchymal cells, approximately 10% myoepithelial cells, and approximately 30% connective tissue cells, including fibroblasts, plasma cells, and microphages. The epithelial cells are characterized by the presence of fat droplets, which in 65% of the cells form large supranuclear vacuoles. Their buoyant density is less than 1.045, allowing their separation from myoepithelial cells and connective tissue cells by isopycnic centrifugation in a density gradient. The homogeneity of the epithelial cell fraction has been assessed by light and electron microscopy. The cells are viable and functionally active as indicated by their ability to exclude vital dyes, incorporate labeled precursors, consume oxygen, maintain intracellular Na+ and K+ concentrations, and retain their structural integrity. In addition, when cultured in Petri dishes, the cells grow as a monolayer, reestablish junctional complexes and retain cell polarity.