OBJECTIVE Pyrimidine antagonist-based chemotherapy is a recommended chemotherapeutic treatment for gestational trophoblastic neoplasia in China. The main reason for treatment failure is chemoresistance. We established a floxuridine (FUDR)-resistant human choriocarcinoma JeG-3/FUDRA sub-line and investigated the role of thymidylate synthase (TS) transcript levels in chemoresistance prediction. STUDY DESIGN The JeG-3/FUDRA sub-line was established by intermittent exposure to increasing doses of FUDR. Levels of TS transcripts were measured by real-time fluorescence quantitative reverse transcription-polymerase chain reaction. beta-hCG secretion in these cell lines has also been detected by using a chemoluminescence method. RESULTS The JeG-3/FUDRA sub-line had a resistance index of 31.62. When exposed to 5.1 x 10(-7) M FUDR, the secretion of beta-hCG was enhanced dramatically, but with the increasing of the FUDR concentration, it was downregulated gradually. According to the concentration of FUDR exposure from low to high, the multiples of free beta-hCG secretion increased 13.19-, 4.76-, 1.90-, 1.44- and 0.47-fold, respectively. The TS transcript level was down-regulated by exposure to a low concentration of FUDR and then gradually increased with increasing doses of the drug. But when a certain concentration was reached, the expression would not increase but decrease sharply. By sub-lines of different concentration of FUDR exposure varying from low to high, the multiples of increases comparing the TS transcript level to parental cells were 0.56-, 0.42-, 1.02-, 2.78- and 2.06-fold, respectively. CONCLUSION We successfully established an FUDR-resistant human choriocarcinoma sub-line and found that the expression of TS mRNA in FUDR-resistant JeG-3 cells was related to the concentration and phase of FUDR exposure. These data suggest that TS mRNA levels would be of limited use as biomarkers for the prediction of chemoresistance to FUDR-based chemotherapy.