We have established and characterized 3 new breastcancer cell lines from pleural effusions of patients with advanced breast cancer. All 3 cell lines, designated IBEP-1, IBEP-2 and IBEP-3, showed typical ultrastructural characteristics of epithelial mammary tumor cells. Electron microscopy showed, among other characteristics, the presence of numerous microvilli, desmosomal junctions, intracytoplasmic duct-like vacuoles, well-developed endoplasmic reticulum and large nuclei. Immunohistochemical and biochemical studies revealed that the 3 cell lines expressed cytokeratin, epithelial membrane antigen, CEA and CA 15-3, but all showed negative immunoreaction for vimentin. On the other hand, other antigens (LEU-M1, GCDFP 15, c-erbB-2) were expressed by some of the cell lines, but in a variable manner. Ploidy studies confirmed the neoplastic origin of the cell lines. The doubling times were 68 hr for IBEP-1, 29 hr for IBEP-2 and 39 hr for IBEP-3. Only IBEP-2 cells expressed estrogen receptors (ER Ψ ), which were down-regulated after preincubation with E 2 , but they did not express progesterone receptors (PgR Ψ ). IBEP-1 and IBEP-3 cells were ER Ψ but expressed PgR (PgR Ψ ). In these 2 cell lines, PgR were downregulated after pre-incubation of the cells with progesterone (10 Ψ8 M) for 24 hr. Estradiol (E 2 ) increased the proliferation rate of IBEP-2 cells and progesterone increased the proliferation of IBEP-1 and -3 cell lines. S.C. injection of the 3 IBEP cell lines into nude mice resulted in the growth of solid tumors between 11 and 16 weeks after inoculation. These cell lines could thus be new models for studying various aspects of the biology and the tumorigenicity of breast-cancer cells. A major interest of these new cell lines is that 2 of them were ER Ψ and PgR Ψ , which is an exceptional phenotypic feature. These 2 cell lines could be interesting models for studying the regulation of PgR and the effects of progestins and antiprogestins independently of the presence of ER.