We investigated 141 bone marrow and 104 venous blood isolates from gastrointestinal cancer patients with a cytokeratin (CK) 20-specific nested reverse transcription PCR for the detection of disseminated tumor cells at time of primary tumor resection. In colorectal cancer patients, 20 of 65 (31%) bone marrow and 9 of 52 (17%) venous blood isolates yielded a CK 20 mRNA-positive result in a stage-dependent manner. The detection rates for gastric cancer patients were 11 of 49 (22%) and 5 of 30 (17%) for bone marrow and venous blood, respectively. In pancreatic cancer patients, positive signals were found in advanced tumor stage. A duplex PCR system improved the feasibility of the test. After analyzing 70 sets of bone marrow and venous blood isolates from colorectal, gastric, and pancreatic cancer patients, we observed a higher detection rate in bone marrow isolates. Survival of patients with CK 20 mRNA-positive findings was significantly shorter than that of negatively tested patients.