MCF-7/ADR cells have been widely used as a multidrug-resistant breast cancer cell model in cancer research. The origin of MCF-7/ADR has been a matter of debate since MCF-7/ADR cells were re-designated NCI/ADR-RES in 1998. Many recent studies still describe MCF-7/ADR cells as originating from the breast cancer cell line MCF-7. Thus, the real origin of MCF-7/ADR cells remains more unclear. In this study, a new adriamycin (ADR)-resistant cell line MCF-7/ADR' was reproduced using the same procedure employed during the initial establishment of MCF-7/ADR. Since the MCF-7/ADR' cell line was definitely derived from parental MCF-7 cells, we were able to directly compare these cell lines together with MCF-7/ADR using immunocytochemical, morphological, and consecutive DNA fingerprinting analyses to determine the true origin of MCF-7/ADR. Both ADR-resistant cell lines displayed some similar phenotypic characteristics, such as high levels of P-glycoprotein (P-gp) expression, increased vacuolation, abundant filamentous material, and irregular pseudopodia. With increasing concentrations of ADR, the DNA fingerprints of MCF-7/ADR' cells were always identical to the parental MCF-7 cells. However, the DNA fingerprints of MCF-7/ADR cells did not relate to MCF-7 or MCF-7/ADR'. MCF-7/ADR and the breast cancer cell line MCF-7 are not of the same origin. Long-time culture in the presence of ADR does not cause significant changes in DNA fingerprint patterns.