Few studies have investigated the biological factors associated with sensitivity to bolus infusions of 5-fluorouracil (5FU), including sequential methotrexate (MTX)/5FU therapy. We investigated the relationship between the expression of thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), E2F-1, Bcl-2, Bak, and Bcl-X, and the chemotherapeutic effects of sequential MTX/5FU. We studied 38 patients with unresectable or recurrent gastric cancer, treated weekly with sequential MTX/5FU (MTX 100 mg/m2, 5FU 600 mg/m2, by bolus infusions, with a three-hour interval). Expression of the above proteins was examined in initial biopsy samples with immunohistochemical methods. Immunohistochemical reactivity was defined as positive when over 25% of cancer cells showed strong staining in the cytoplasm for TS, TP, DPD, Bak, Bcl-2, and Bcl-X, and in the nucleus for E2F-1. The overall response rate was 28% in the 29 patients who had measurable lesions. Bak-negative patients showed a higher response rate than Bak-positive patients (39% versus 9%, respectively; p=0.1096), although expression of the other proteins was not associated with chemosensitivity. The median survival time (MST) of all patients was 256 days. Bak-negative patients survived significantly longer than Bak-positive patients (MST, 302 days versus 134 days, respectively; p=0.0044). Bcl-X-negative patients survived significantly longer than Bcl-X-positive patients (MST, 302 days versus 215 days, respectively; p=0.0080). Furthermore, patients negative for both Bak and Bcl-X had significantly better prognoses than other patients (MST, 373 days; p<0.0001). Within the limits of the small patient population, multivariate analysis using the Cox proportional hazards model showed that Bak, Bcl-X, and histological type were independent variables predicting survival (p=0.0008, 0.0081, and 0.0082, respectively). Although previously described predictive markers for protracted infusion of 5FU, including TS, TP, and DPD, might not be associated with clinical outcome in patients treated with sequential MTX/5FU, Bak may be a useful marker for chemoresponse and survival. Furthermore, both Bcl-X expression and the coupled expression of Bak and Bcl-X, as well as histological type, may be useful predictive markers for survival.