We have used clonogenic survival assays and flow cytometry of human lung A549, breast MCF7 and pancreas adenocarcinoma P-SW cell lines to examine the effects of gemcitabine (2'-deoxy-2', 2'-difluorocytidine) in combination with cisplatin, paclitaxel or radiation. Additive cell killing was observed for all cell lines when they were treated with cisplatin for 1 h followed by varying concentrations of gemcitabine for 24 h. Likewise, additive cell killing was noted in all cell lines when treated with gemcitabine for 24 h followed by varying doses of radiation. When A549 cells were exposed to gemcitabine for 24 h followed by a 1 h exposure to cisplatin, synergistic effects were noted. Using the latter regimen, MCF7 cells demonstrated additive cell kill, while the P-SW cells showed a more complex relationship with additive killing below 50 nM gemcitabine and less than additive effect above 50 nM gemcitabine. All three cell lines were also tested with various gemcitabine/paclitaxel combinations. When gemcitabine and paclitaxel were incubated concurrently, gemcitabine antagonized the cell kill produced by paclitaxel. All cell lines showed less than additive killing when either gemcitabine incubation preceded the paclitaxel incubation or the paclitaxel incubation preceded the gemcitabine incubation. Our results show that gemcitabine acts as a radiation sensitizer to increase the effects of radiation. Likewise, we demonstrate that the only uniformly beneficial drug combination schedule in all three cell lines was when cisplatin incubation preceded gemcitabine incubation. The gemcitabine/paclitaxel combinations were much more disturbing with respect to potential clinical trials. Our results would caution any planned clinical trials combining paclitaxel with gemcitabine to be reconsidered because of the potential for less than additive and even antagonistic effects of the combination.