The anticancer agent doxorubicin has two different effects on human SK-MEL-170 melanoma cells: the well known direct cytotoxicity and a marked enhancement of their susceptibility to killing by the R24 monoclonal anti-GD3) ganglioside antibody and human complement. This complement-enhancing effect of doxorubicin is also present after covalent immobilization onto glycerol-coated glass beads preventing cellular uptake of the drug (M. Panneerselvam, R. Bredehorst, and C-W. Vogel, Proc. Natl. Acad. Sci. USA, 83: 9144-9148, 1986). In order to investigate the effect of doxorubicin resistance on the complement-enhancing activity of the drug, we have established a doxorubicin-resistant SK-MEL-170 subline. The development of drug resistance in these melanoma cells was associated with multiple phenotypical changes including an increased expression of at least four high molecular weight plasma membrane proteins or glycoproteins with molecular weights of approximately 220,000, 180,000, 150,000, and 130,000, respectively. The drug-resistant cells accumulated doxorubicin at approximately 2-fold lower amounts in accordance with a 2-fold higher efflux of doxorubicin from these cells. The basal complement susceptibility of the doxorubicin-resistant cells was reduced by more than 60% presumably as a result of the observed reduced expression of GD3 sites and decreased binding of C3b. Most importantly, the doxorubicin-resistant cells were also resistant against the complement-enhancing effect of the free and immobilized drug. The two doxorubicin resistance phenomena, the resistance to the cytotoxic and to the complement-enhancing activities of the drug, seem to be fundamentally different: (a) to achieve comparable cytotoxic and complement-enhancing effects on drug-sensitive and -resistant SK-MEL-170 cells, the drug-resistant cells required 178-fold higher concentrations of free doxorubicin for the cytotoxic effect but only 5-fold higher amounts of the free drug and 12-fold higher amounts of the immobilized drug for the complement-enhancing effect; (b) resistance against the cytotoxic activity of doxorubicin is associated with reduced intracellular drug accumulation, while the data obtained with immobilized doxorubicin indicate that resistance to the complement-enhancing activity of the drug is independent of cellular drug uptake. These data suggest that different mechanisms are responsible for the two resistance phenomena in doxorubicin-resistant melanoma cells.