Orotate phosphoribosyltransferase (OPRT, EC 2.4.2.10), a key enzyme that catalyzes one of the primary first-step phosphorylation processes of fluoropyrimidine, has recently been recognized as an important factor that primarily determines the anticancer effects of S-1. The OPRT levels were examined in 97 gastric carcinoma tissues and 65 normal gastric mucosa tissues obtained from patients with gastric carcinoma using a newly-developed enzyme-linked immunosorbent assay. Correlations with thymidylate synthase and dihydropyrimidine dehydrogenase levels and the effects of neoadjuvant chemotherapy were evaluated. The OPRT level in gastric carcinoma tissue was significantly higher than that in normal gastric mucosa. There was no correlation of OPRT level with either TS or DPD levels. There was no correlation of OPRT level between those in gastric carcinoma and those in normal gastric mucosa simultaneously obtained from identical patients. The OPRT levels in patients who underwent neoadjuvant chemotherapy were not different from those without neoadjuvant chemotherapy. These results suggest that activation of fluoropyrimidine mainly occurs in carcinoma tissues and the OPRT levels in carcinoma tissues were not influenced by neoadjuvant chemotherapy with fluoropyrimidine.