The methotrexate analog edatrexate has been shown to have greater antitumor activity and an improved therapeutic index as compared with its parent compound in preclinical systems. These studies suggest that edatrexate may have a broad role in the treatment of solid tumors. Information regarding edatrexate in combination with other chemotherapeutic agents is limited. We evaluated the interaction of edatrexate with cisplatin in vitro as assessed by median-effect analysis in the A549 human lung-cancer cell line. The effects of dose, exposure time, and schedule dependence were assessed. Cytotoxicity was evaluated using the tetrazolium-based colorimetric (MTT) assay. The inhibitory concentration producing 50% absorbance (IC50 for edatrexate with 1 h exposure was 1.4 microM. For all combination experiments, the edatrexate dose was fixed at 0.2 microM (IC10) whereas cisplatin (CDDP) concentrations were varied for 1-, 3-, and 24-h exposures either before or after edatrexate treatment. Drug interactions were assessed using the combination-index method as defined by median-effect analysis. A synergistic interaction was documented in experiments when edatrexate was applied prior to CDDP (combination index, < 1). The combination studies in which edatrexate was used prior to CDDP resulted in significant reduction of all three CDDP IC50 values: 1-h IC50, from 30.0 to 3.9 microM; 3-h IC50, from 21.3 to 1.4 microM; and 24-h IC50, from 1.7 to 0.03 microM. In contrast, synergism was not observed in experiments in which edatrexate treatment occurred after cisplatin exposure. Median-effect analysis is a useful method of determining drug interactions. In the present study, the combination of edatrexate and CDDP demonstrated schedule-dependent synergism, with the synergism being observed only in the setting of edatrexate treatment before CDDP exposure. Due to the potential broad spectrum of activity of edatrexate plus CDDP, further studies are warranted to determine the mechanism responsible for the synergism and to investigate this combination in a variety of tumor models.