The resistance of cancer cells to multiple chemotherapeutic agents poses a major problem in the successful treatment of breast cancer. Whether drug resistance is due to changes induced in the drug-exposed tumor cells or represents the selective growth of one or more drug-resistant clones present in the initial tumor remains controversial. Here we provide evidence that the development of multidrug resistance in a human breast cancer cell line (MCF-7) is a result of propagation of an inherently resistant subclone. The drug-resistant MCF-7 (MCF-7/DOX) cells exhibited several phenotypic and genotypic features that were notably distinct from those observed in the parental drug-sensitive (MCF-7/WT) cells. The most striking change was the presence of a full-length functional caspase-3 in MCF-7/DOX cells that was missing in the parental MCF-7/WT cells due to a deletion mutation in the caspase-3 gene. A drug-resistant MCF-7 cell subline (MCF-7/WT/DOX) was established by exposing the MCF-7/WT cells directly to a high dose of doxorubicin and used for determining the phenotypic and genotypic alterations associated with drug resistance. The MCF-7/WT/DOX cells exhibited alterations identical to those of the MCF-7/DOX cells but which were strikingly distinct from the parental MCF-7/WT cell line. These results suggest that drug resistance is an inherent property of some cancer cells that are present in the initial tumor burden and exhibit distinct phenotypic/genotypic alterations.