Four human colon cancer cell lines (SW620, LS 180, DLD-I, and HCT-15) and Adriamycin-resistant sub-lines with varying degrees of P-glycoprotein expression were studied to evaluate the reversibility of Adriamycin resistance in human colon cancer. Two groups of cell lines were studied. In the first, including a series of Adriamycin-resistant SW620 and DLD-I sub-lines, and in parental HCT-15 cells, P-glycoprotein has a major role in Adriamycin resistance, as evidenced by a correlation between Adriamycin resistance, expression of the multidrug-resistance gene mdr-I and its product, P-glycoprotein (Pgp), decreased drug accumulation and reversibility by verapamil. In these cell lines, increasing doses of verapamil are required to fully reverse increasing levels of resistance. In the second group, including parental SW620, DLD-I and LS 180 cells and Adriamycin-selected LS 180 sub-lines, P-glycoprotein does not have a major role in Adriamycin resistance. There was correlation between the schedule dependence of Adriamycin cytotoxicity and the role of P-glycoprotein in modulating resistance. In the cell lines in which P-glycoprotein was a major determinant of Adriamycin resistance, the drug exposure (defined as the product of the concentration and the time of treatment) needed to achieve a given percent cell kill was reduced as much as 9-fold when cells were treated for 7 days as compared with 3 hr. By comparison, in cell lines in which P-glycoprotein played a lesser role, the drug exposure necessary to achieve a given percent kill increased under conditions of continuous treatment. In some human colon carcinoma cell lines Pgp appears to play a significant role in resistance to Adriamycin, and this can be overcome by the use of competitive inhibitors of Pgp. The increased sensitivity with continuous treatment observed in cell lines with P-glycoprotein-mediated resistance suggests that administration of drugs by continuous infusion may be valuable in reversing clinical drug resistance mediated predominantly by P-glycoprotein.