Little is known about the expression levels of thymidylate synthase (TS), thymidine phosphorylase (TP), dihydropyrimidine dehydrogenase (DPD), and orotate phosphoribosyltransferase (OPRT) in diffusely infiltrating colorectal cancer. We semiquantified the mRNA levels of these enzymes in colorectal cancer specimens from 10 patients with diffusely infiltrating type and 20 patients with other types (control) matched by the maximal diameter and pathological stage, using the Dannenberg tumor profiling method. The relative expression of these enzymes did not significantly differ between diffusely infiltrating and other types of tumors. There were no significant relationships among the relative expression of the four enzymes in the diffusely infiltrating tumors, while the following three combinations were found to be correlated with each other in the control tumors: TS versus TP,TS versus DPD, and TP versus DPD. In 5 patients who received 5-fluorouracil-based chemotherapy for evaluable lesions, there were no specific relationships between the expression of these enzymes and therapeutic response. In conclusion, diffusely infiltrating colorectal cancer does not appear to have any characteristics in terms of the expression of these enzymes, which may not alter the effect of 5-fluorouracil-based chemotherapy.