Five human melanoma cell lines (C8146C, C8161, C82-7A, C83-2CY and MIRW5) were shown to contain a significant number of melanoma colony-forming units resistant to single-agent treatment by dexamethasone, alpha-interferon and trans-retinoic acid. These biological modifiers were combined with difluoromethylornithine into a low-dose combination using concentrations below pharmacologically achievable levels. The suppression of melanoma colony formation induced by this combination was consistent and significantly higher than that seen with any single agent, colony formation being reduced by an average of 90%. Leaving either DEX or DFMO out of the 4-agent combination resulted in a significant decrease in the observed inhibition. This was also verified by the addition of putrescine which inhibited only the DFMO activity. Median effect analysis of the DFMO + IFN inhibition of C8161 cells demonstrated that the 2 agents interacted synergistically over the entire dose-response curve. Of the high-dose combination-treated melanoma colony-forming units, 97% did not form small growth units; most remained as arrested single cells, but the cells and small growth units could still metabolize tetrazolium stain after the experiment, suggesting that the high-dose combination arrested the growth of the melanoma colony-forming units via a non-cytotoxic mechanism.