Aim: S-1 is an oral anticancer fluoropyrimidine formulation consisting of tegafur, 5-chloro-2,4-dihydroxypyridine and potassium oxonate. The aim of this study was to evaluate the pharmacokinetics and bioequivalence of a newly developed generic formulation of S-1 in Chinese cancer patients in comparison with the branded reference formulation of S-1. Methods: A single-dose, randomized-sequence, open-label, two-way self-crossover study was conducted in 30 Chinese cancer patients. The subjects alternatively received the two formulations (40 mg/m(2), po) with a 7-d interval. Plasma concentrations of FT, CDHP, Oxo, and 5-Fu were determined using LC-MS/MS. Pharmacokinetic parameters, including C-max, T-max, t(1/2), AUC(0-t), and AUC(0-infinity) were determined using non-compartmental models with DAS2.0 software. Bioequivalence of the two formulations were to be evaluated according to 90% CIs for the log-transformed ratios of AUC and C-max of S-1. Adverse events were evaluated through monitoring the symptom, physical and laboratory examinations, ECGs and subject interviews. Results: The mean values of C-max, AUC(0-t), and AUC(0-infinity) of FT, 5-Fu, CDHP, and Oxo for the two formulations had no significant differences. The 90% CIs for natural log-transformed ratios of C-max, AUC(0-t), and AUC(0-infinity) were within the predetermined bioequivalence acceptance limits. A total of 11 mild adverse events, including fatigue, nausea and vomiting, anorexia, diarrhea and myelosuppression, were observed, and no serious and special adverse events were found. Conclusion: The newly developed generic formulation and reference formulation of S-1 have similar pharmacokinetics with one dose (40 mg/m(2)) in Chinese cancer patients. Both the formulations of S-1 are well tolerated.