The antitumor efficacy of the combination of nedaplatin (NDP) with gemcitabine (GEM) was evaluated against Ma44/GEM, a GEM-refractory subline of Ma44 human lung cancer, which was established by serial in vitro passage of Ma44 cells in the presence of GEM.Ma44/GEM showed less sensitivity to GEM and cytosine arabinoside with resistance factors of 7.7 and 8.3, respectively, but not to Taxol, Irinotecan, Mitomycin C and NDP. Flow cytometry analysis demonstrated that membrane transporter molecules such as multidrug-resistant, multidrug-resistant related protein or lung resistant protein were not induced in Ma44/GEM cells. In vivo experiments confirmed the less sensitivity of Ma44/GEM to GEM. The resistant factor of Ma44/GEM to GEM in vivo was estimated to be 6.7 in terms of ED(50).MA44/GEM-implanted athymic mice were treated with GEM i.v. once followed by i.v. injection of NDP at an interval of approximately 30 min. The mice were treated again with GEM after 3 or 4 days. The combined dosing of NDP with GEM resulted in synergistically enhanced inhibition of tumor growth against Ma44/GEM. The antitumor efficacy of the combination of NDP and GEM was superior to the best effect of either monotherapy. These results demonstrate the effectiveness of the combination of NDP with GEM against the GEM-refractory human lung cancer model.