In colon cancer the presence of metastases in the regional lymph nodes is an important prognostic factor. Currently, examination is based on histological and immunohistochemical evaluations of lymph node sections. However, these methods are time-consuming, labour intensive and micro-metastases might be missed. The sentinel node technique may be used to identify the direct tumour draining lymph node. In this study the possible use of flow cytometry for detection of sentinel node metastases in patients with colon cancer has been investigated. Peripheral blood mononuclear cells (PBMC) with the addition of DLD-1 colon cancer cells were stained intracellularly for cytokeratin 20 (CK20), and for the cell surface markers epithelial cell adhesion molecule (EpCAM) and carbohydrate antigen 19-9 (CA19-9). CK20 positive colon cancer cells were reliably detected with as few as 0.037% events. However, PBMCs from both colon cancer patients and from healthy individuals contained CK20 positive cells. Staining for EpCAM resulted in an almost complete recovery of tumour cells, and as few as 0.037% added cells were detected. Low intra-assay variability was determined for EpCAM (CV 8.8) and CA19-9 (CV 9.7) stainings. The results from staining for CK20 or for EpCAM and CA19-9 concorded, but the degree to which respective antigens were expressed varied. The use of flow cytometry for detection of metastatic colon cancer cells was verified in fourteen sentinel nodes specimens from patients with colon cancer. Herein we have explored the potential of flow cytometry to become a fast, sensitive and reliable method for detection of lymphatic metastases in patients with colon cancer using direct fluorophore-conjugated antibodies against multiple surface antigens. The method seems feasible and further testing is warranted.