We assessed the antiproliferative effects of tumor necrosis factor (TNF-alpha) and gamma-interferon (IFN-gamma) alone and in combination, on nine human colon carcinoma cell lines. All were resistant (less than 30% inhibition) to TNF-alpha alone. Four cell lines were resistant to IFN-gamma alone, two exhibited a minimal degree of sensitivity (30-50% inhibition), one was moderately sensitive, and two were inhibited 70% or greater. A synergistic antiproliferative effect occurred in eight of the nine cell lines treated with a combination of TNF-alpha and IFN-gamma. In seven of these eight, the combination of cytokines resulted in 30-40% more growth inhibition than predicted had an additive interaction occurred (P less than 0.005). In two cell lines with an induced resistance to mitomycin C, an increase in resistance to combined TNF-alpha and IFN-gamma treatment correlated with an increasing resistance to mitomycin C. The data were further analyzed to determine if combination treatment altered the sensitivity of the cells to one or both agents in addition to synergistically potentiating growth inhibitory effects. Combinations of TNF-alpha/IFN-gamma enhanced the dose response activity of TNF-alpha in three cell lines (P less than or equal to 0.09) and decreased the dose response activity of IFN-gamma in another three (P less than or equal to 0.02). Colony forming experiments on HCT 116 cells demonstrated a reduction in the number of 250-micron colonies in the IFN-gamma/TNF-alpha treatment groups when compared to controls, indicating that combined treatment had a cytotoxic effect. We conclude that combination TNF-alpha/IFN-gamma treatment has a synergistic cytotoxic effect on human colon carcinoma cells. IFN-gamma may enhance the effectiveness of TNF-alpha in some cell lines, but not conversely. These results may have therapeutic implications.