A series of doxorubicin-resistant variants of the human LS I74T colon carcinoma cell line was generated by stepwise selection. These variants also exhibited increased resistance to vinblastine, etoposide, cis-platinum, and melphalan, suggesting that resistance was multifactorial. The parental LS I 7 4 1 cell line and 3 resistant variants were examined for over-expression of P-glycoprotein, changes in total cellular glutathione content, and the level of topoisomerase-ll expression. Changes in all of these parameters were observed in the doxorubicin-selectants, along with a marked shift in the intracellular distribution of doxorubicin. P-glycoprotein RNA and protein levels were increased 2to 3-fold in the resistant variants, while total glutathione levels increased I .4-to 2. I -fold. Treatment with DL-buthionine-[S,R]-sulfoximine, an inhibitor of glutathione biosynthesis, was able to reverse resistance to cis-platinum and melphalan in these variants, but had little effect on doxorubicin resistance. lmmunoblot analysis of cell extracts indicated that the level of DNA topoisomerase II (EC 5.99. I .3) in the doxorubicin-resistant LS I74T cells was decreased by approximately 50% compared with the parental cell line. Doxorubicin was mainly localized to the cytoplasm in resistant cells, while in the parent line it was mostly found in the nucleus. This constellation of changes suggests that selection with doxorubicin activated several mechanisms of resistance involving drug transport, metabolism, and ability to reach nuclear target sites.