Orotate phosphoribosyltransferase (OPRT) is an enzyme that causes the activation of 5-fluorouracil (5-FU). Dihydropyrimidine dehydrogenase (DPD) is known to catabolize 5-FU, which is widely used in chemotherapeutic treatments for patients with a variety of malignant tumors including gastric and colorectal cancer. The expression and activities of these two enzymes therefore play important roles in the response of cancer patients to chemotherapy. However, little is known about the expression of these enzymes in gastric cancer. In the present study, we further elucidate the expression patterns of ORPT and DPD and their clinicopathological significance by immunohistochemical analysis in 221 and RT-PCR in 36 gastric cancer samples. The expression of OPRT by immunohistochemical analysis was detected in 117 (52.9%) cases, whereas DPD was detected in 66 (29.9%) cases. Moreover, the level of expression of OPRT was found to correlate with the depth of tumor invasion and a poorer prognosis. Although the mRNA and protein expression of OPRT and DPD levels did not correlate, an inverse correlation in the expression of OPRT and DPD was observed by RT-PCR. The survival benefit of post-operative adjuvant chemotherapy could not be confirmed in our present analysis. However, among the patients who had received such treatment with 5-FU or its derivatives, the prognosis in cases with low DPD levels was better than that in cases with high DPD expression by immunohistochemical analysis. These results indicate that the expression of OPRT and DPD are important predictors of both survival and the response to adjuvant chemotherapy in gastric cancer patients.