The antiproliferative effect of 5-fluorouracil (5-FU) in colon cancer can be enhanced by interferons (IFN-alpha and IFN-gamma). The mechanisms by which IFNs modulate 5-FU activity are not completely elucidated. IFN-alpha may elevate the levels of the active 5-FU metabolite 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) in the cell, possibly leading to increased inhibition of the target enzyme thymidylate synthase (TS), which might enhance DNA damage. It has been shown that IFN-gamma can prevent 5-FU induced overexpression of TS. We studied IFN modulation in three colon cancer cell lines (SW948, WiDr, human; C26-10, murine) and the sublines WiDr/F and C26-10/F, which were adapted to low folate levels. A 1.5-fold increase in 5-FU sensitivity was observed in C26-10 and C26-10/F (by murine IFN-alpha, beta); in SW948, WiDr and WiDr/F (by human IFN-gamma) and in SW948 and WiDr/ F (by human IFN-alpha). In none of the cell lines did human IFN-alpha, IFN-gamma or murine IFN-alpha, beta increase FdUMP levels after exposure to 5-FU. TS activity, indirectly measured by incorporation of [6-3H]-deoxyuridine into DNA, was inhibited by 5-FU, but the IFNs did not enhance inhibition. DNA damage was measured as a drug-induced decrease of double-stranded (dss) DNA compared to control cells. After 5-FU exposure, dss DNA decreased to 60-75% in WiDr, WiDr/F and SW948 cells. Human IFN-alpha alone caused minimal DNA damage (95% dss DNA), but increased 5-FU-induced effects to 35-50% dss DNA. IFN-gamma did not cause DNA damage and did not enhance 5-FU-mediated DNA damage. Expression of TS protein, analysed by ELISA, was increased after 5-FU exposure of SW948 cells, but this increase was not affected by addition of either IFN-alpha or IFN-gamma. It is concluded that one of the mechanisms involved in modulation of 5-FU activity is the effect of IFN-alpha on 5-FU-mediated DNA damage, but for IFN-gamma no mechanism of action was found.