Cytotoxicity studies combining edatrexate (EDX) and cisplatin (Cis-Pt) were carried out in HL-60 cells in vitro as a retrospective analysis of the same combination in animal models and as a prospective study of this combination for future clinical trials. For purposes of comparison, parallel studies were carried out using methotrexate (MTX) and Cis-Pt. Dose-effect relationships were analyzed by the median-effect principle and the combination index-isobologram technique. EDX was the most cytotoxic agent of the three examined. The doses effective in 50% inhibition of the cell proliferation (ED50 values) for EDX, MTX, and Cis-Pt were 0.001, 0.0043, and 1.08 microM, respectively. Synergism occurred at effect levels corresponding to greater than 65% inhibition of cell growth by EDX + Cis-Pt, with an increase in synergism being observed at high doses. By contrast, MTX + Cis-Pt exhibited moderate synergism, with a decrease in synergism being noted at high doses. Preceding one drug by another for 4 h during the 48-h incubation period did not result in synergism greater than that produced by simultaneous exposure to both drugs for both pairs of combinations. Due to the synergism arising from these combinations, the ED90 values can be reduced by as many as 52 and 7.3 times for Cis-Pt and EDX, respectively, as compared with only 4.0 and 1.9 times for Cis-Pt and MTX, respectively. The calculation of these drug interactions was carried out automatically with the use of computer software and was also illustrated by a sample calculation performed without computer simulation.