NAD(P)H:quinone oxidoreductase (DT-diaphorase; DTD) plays a major role in activating mitomycin C (MMC) in human colon and gastric carcinoma cell lines. Thus, measurement of DTD in clinical tumor samples could be beneficial in designing adjuvant chemotherapy. We explored immunological quantitation of DTD protein using a monoclonal antibody against DTD, demonstrating a close correlation between protein expression and enzyme activity of DTD in colon and gastric carcinoma cell lines and in colorectal tumor samples. This indicates that such immunoblot analysis is a simple alternative method for quantitating DTD in clinically excised samples. In most colorectal tumor samples, the tumors expressed larger amounts of DTD than did the peripheral normal tissues, suggesting a selective toxicity of MMC toward tumor cells. Also tumors with nodal metastases showed significantly higher DTD levels than did tumors without metastasis. These results raise the possibility that DTD expression is related to tumorigenesis and malignant progression of colorectal tumors. Measurement of DTD by the immunological method described here could be beneficial in designing a rational adjuvant chemotherapy with MMC.