TCF transcription factors are mediators of the WNT signaling pathway and are antagonized by the transforming growth factor beta signaling pathway. Here human TCF-3 has been cloned and characterized. Differential expression analyses of TCF genes in gastric cancer revealed that TCF-1 was expressed in most cases of primary gastric cancer at almost the same level as in normal gastric mucosa and that TCF-3 was occasionally up-regulated in primary gastric cancer. The TCF-3 expression vector was transfected to gastric cancer cell line MKN28 to establish stable transformants. Three independent MKN28 transformants overexpressing TCF-3 showed about 8-fold resistance to mitomycin C (MMC; IC50, 2.4 microg/ml) compared with MKN28 vector transfectants (IC50 = 0.3 microg/ml). Among the 10 drug resistance-associated genes examined in this study, the DT-diaphorase (DTD) gene was down-regulated in three MKN28 transformants overexpressing TCF-3. DTD mRNA was also down-regulated in primary gastric cancer with TCF-3 up-regulation. In addition, DTD protein was down-regulated in three MKN28 transformants overexpressing TCF-3 compared with MKN28 vector transfectants. DTD is implicated in the activation of MMC in target cells, and DTD down-regulation explains MMC resistance. MMC resistance induced by TCF-3 overexpression is probably due to DTD down-regulation, which might provide a possible target for new therapy of drug-resistant gastric cancer.