The significance of tumor tissue thymidine phosphorylase (TP) and dihydropyrimidine dehydrogenase (DPD) levels, as well as the TP/DPD ratio have recently been reported as prognostic factors and for custom-made chemotherapy. However, there have been no distinct studies on actual tumor sampling methods. For 16 patients who had undergone resection of advanced colorectal cancer, we: i) measured TP and DPD levels in different portions of the tumor using enzyme-linked immunosorbent assay (ELISA); ii) categorized the tumor into an edge, center, and base area, and histo-pathologically calculated the ratio of cancer cell/cancer cell + stromal cell; and iii) examined the correlation between cancer and stromal cell TP expression and TP value. Variation within the same tumor was seen in each activity level and TP/DPD ratio. The ratio of cancer cell in the edge area was high, with the ratio of stromal cell in the center and base areas increasing in that order. A correlation was seen between TP expression and TP levels, and TP expression was evident in the stromal cells. It is therefore recommended to sample the edge area for tumor TP levels.