Previous in vitro and in vivo studies have shown a synergism between interferon (IFN) and 5-fluorouracil (5-FU) against different tumor cell lines. In the present study we report that the combination of IFN-alpha and 5-FU has a significant effect not only on the inhibition of tumor cell growth but also on the regulation of natural killer cell-mediated cytotoxicity (NK-CMC). The addition of 5-FU to effector cell population neither affects NK cell activity nor activation of NK cells by IFN or by interleukin (IL)-2. However, pretreatment of target cells with 5-FU increased their susceptibility to NK activity and abolished the protective effect induced by IFN against NK-CMC. This dual effect of IFN-alpha and 5-FU was found to be applicable to target cells of different origins including a cervical carcinoma cell line (ME-180), a hairy cell leukemia-like cell line (Eskol), a CML cell line (K-562) and a primary culture of AIDS-related Kaposi's sarcoma cells. Similar results were found with IL-2 treatment of Eskol cells but not other cells. Combination of IL-2 with 5-FU resulted in enhancement of the sensitivity of the cells to NK activity and abolished the protection against NK-CMC. Based on these results we propose that the combination of IFN-alpha and 5-FU not only has a direct growth inhibitory effect on tumor cells but also has a regulatory role on the immunological arm of the NK-CMC. Moreover, since the combination gave the same pattern of response in different tumor cells, both NK-sensitive and NK-resistant, this combination treatment may be a candidate for clinical trials in various types of tumors.