Most of the available human breast tumor cell lines have been derived from pleural effusions. The two cell lines herein described, BT-474 and BT-483, were derived from solid, invasive ductal breast carcinomas. Both are epithelial and neoplastic as judged by their general morphology, their fine structure, and their ability to produce growing nodules in nude mice and colonies in soft agar and methocel. BT-474 and BT-483 are human as expressed by chromosome morphology and aneuploid with a modal number of 55 and 72 chromosomes, respectively. Trypsin-Giemsa banding did not reveal the presence of obvious HeLa markers, and the glucose 6-phosphate dehydrogenase electrophoretic migration pattern was of the B-type. Furthermore, the migration of lactic dehydrogenase, malic dehydrogenase, and 6-phosphogluconate dehydrogenase isoenzymes was consistent with a human pattern and different from that of the mouse, rat, or hamster. Quarterly tests to detect the presence of aerobic and anaerobic mycoplasmas were repeatedly negative. A culture medium containing insulin, increased amounts of amino acids, vitamins, and glucose facilitated the isolation of the tumor cells. Cell replication was maintained with 10% fetal calf serum absorbed with activated charcoal and dextran. No production of alpha-lactalbumin was detected by radioimmunoassays, but high levels of progesterone receptors were found in both cell lines.