Four human breast cancer cell lines with or without estrogen and progesterone receptors were adapted to growth in the continuous presence of doxorubicin (Dox) at 10 (Zr-75-1), 15 (HTB-122), or 50 (MDA-MB-231 and Hs578T) ng/ml. The sublines of Zr-75-1, MDA-MB-231 and Hs578T showed 5-10-fold Dox resistance and also cross-resistance to vincristine (VCR) and etoposide (VP16). The sublines of Zr-75-1, MDA-MB-231 and Hs578T showed 5-10-fold Dox resistance and also cross-resistance to vincristine (VCR) and etoposide (VP16). The sublines maintained or slightly increased their cis-platinum (CDDP) sensitivity. The sublines of HTB-122 showed resistance only to VP16 combined with a paradoxical increased sensitivity to VCR. The phenotypic alteration in the sublines with respect to Dox sensitivity was maintained for at least two months in the absence of Dox. The glutathione depletor buthionine sulfoximine (BSO) and the calcium channel blocker verapamil (Ver) increased the Dox sensitivity slightly only in the MDA-MB-231 and Hs578T sublines, respectively. Ver also tended to protect some of the sublines from CDDP. The sublines of Zr-75-1 and Hs578T showed increased expression of the 170-kDa permeability glycoprotein (P-gp), whereas expression of a 85-kDa membrane protein determined by the MRK20 antibody was increased in the sublines of Zr-75-1, and HTB-122. Class pi glutathione transferase (GST) levels varied greatly between the cell lines but increased during Dox selection only in the subline of Zr-75-1. Class mu GST was detectable in the MDA-MB-231, Hs578T and HTB-122 cell lines, whereas class alpha GST was detectable in these sublines but undetectable in their parental cell lines. The Zr-75-1 subline showed a 5-fold increase in the class alpha concentration. Except for a correlation between increased P-gp expression and resistance to Dox, VCR and VP16, no obvious correlations between receptor status, increased P-gp expression, membrane proteins, GST levels and acquired drug resistance were found. Thus, except for a possible role for P-gp in multidrug-resistance, these findings indicate a pronounced mechanistic heterogeneity responsible for cytotoxic drug sensitivity also in cells with a common histologic origin and exposed to the same drug.