Gamma-Interferon (IFN-gamma) and the antimetabolites 5-fluorouracil (5-FU) and 5-fluoro-2'-deoxyuridine (FUdR) were investigated as individual agents and in combination for their in vitro antiproliferative capacity and for their effect on the expression of HLA class-I antigen, carcinoembryonic antigen (CEA) and the intracellular tumor-associated antigen CTA-I in 7 human colorectal cancer cell lines: WiDr, HT29, Colo 205, SW116, LS174T, SW1398, and LoVo. Growth inhibition by IFN-gamma at clinically relevant concentrations (50-100 U/ml) was found in 4/7 cell lines. The cell lines were equally sensitive to 5-FU (IC50 in a range of 2-10 microM), while sensitivity to FUdR varied considerably (IC50 in a range of 0.01-90 microM). When 50 U/ml IFN-gamma were combined with 5-FU or FUdR, the antiproliferative effects were synergistic in those cell lines with sensitivity to IFN-gamma as a single agent, but not in the IFN-gamma-insensitive cell lines. IFN-gamma was able to enhance the expression of HLA Class I and CEA in 4/7 and 3/7 cell lines, respectively, as measured by flow cytometry. CTA-I expression could not be enhanced with IFN-gamma. The expression of the 3 antigens tested was also increased by 5-FU and FUdR. This effect was concentration-dependent in most instances and varied between the individual cell lines. The combination of 50 U/ml IFN-gamma with 25% growth-inhibitory concentration of 5-FU or FUdR for each cell line resulted in an additional increase in antigen expression in 4/7 cell lines. No relation was found between the enhancement of antigen expression and the sensitivity to IFN-gamma or the anti-metabolites. The enhancement in antigen expression also did not show a relationship with changes in cell-cycle distribution upon exposure to IFN-gamma or the anti-metabolites. These results suggest independent mechanisms for the antiproliferative and antigen-enhancing effects of IFN-gamma, 5-FU and FUdR.