AbstractMCF‐10A human mammary epithelial cells cultured inside reconstituted basement membrane form acini that resemble the acinar structures of mammary lobules. This three‐dimensional culture system has been used for identifying and characterizing the signal transduction pathways controlling cell proliferation and death, and for studying their disregulation in malignant progression. We have compared the ultrastructure of MCF‐10A acini, MCF‐10A cells grown in monolayer, and the acinar structures of human breast lobules. The tissue architecture of MCF‐10A acini was formed by hemidesmosomes connected to a basement membrane and by abundant desmosomes between acinar cells. Intermediate filaments that joined into large and abundant filament bundles connected hemidesmosomes and desmosomes to sites at the nuclear surface. Fewer and thinner bundles of filaments were observed in monolayer MCF‐10A cells and even fewer in breast tissue. Tight junctions were observed between cells in breast tissue but missing in MCF‐10A acini. The cytoplasm of MCF‐10A acinar cells had a polar organization similar to that observed in breast tissue, with centrosomes and the Golgi apparatus on the apical side of the nucleus. MCF‐10A acinar nuclei had an irregular, frequently invaginated surface and had a single nucleolus. The distribution of heterochromatin was similar to that in the epithelial cells of breast tissue. The nuclei of monolayer MCF‐10A cells had multiple nucleoli, a more regular profile, and less heterochromatin. Electron microscopy has the resolution required to survey features of MCF‐10A cell and acinus architecture that may change with manipulations designed to induce malignant phenotypes. J. Cell. Physiol. © 2006 Wiley‐Liss, Inc.