Both cisplatin (CDDP) and leucovorin (LV) have been shown to enhance cytotoxicity of 5-fluorouracil (FUra) against murine and human neoplasms by increasing intracellular reduced folate concentrations. We were interested in their use in a combination to inhibit non-small cell lung cancer (NSCLC) cell growth and therefore conducted an in vitro study to investigate the cytotoxic activities of combinations of CDDP plus FUra, with and without LV (20 microM), against seven NSCLC cell lines. A tetrazolium assay with application of the classical isobole method was used to test drug combinations. We found that LV enhanced FUra but not CDDP cytotoxicity and that the degree of enhancement was negatively correlated with the effect of FUra. There was an overall additive combination effect of CDDP plus FUra, although there may be synergy at higher effect levels. There was synergy to a combination of CDDP, FUra, and LV, presumably primarily related to the synergistic effects of adding LV to FUra. In summary, LV and CDDP enhanced FUra cytotoxicity in a complementary fashion and there was clear synergy of a combination of CDDP, FUra, and LV against a panel of NSCLC cell lines. Our in vitro results provide a rationale for controlled clinical studies of this three-drug regimen in patients with NSCLC.