About 20% to 30% of colon cancer patients classified as node negative by routine hematoxylin-eosin (H&E) staining are found to have micrometastases (MM) or isolated tumor cells (ITC) in sentinel lymph nodes (SLNs) if analyzed by step sections and immunohistochemistry (IHC). Whether SLNs are in this respect representative for all lymph nodes was addressed in this study. SLNs were identified using the intraoperative blue dye detection technique. If all lymph nodes (SLNs and non-SLNs) of a patient were negative by routine H&E staining, they were step-sectioned and analyzed by IHC using pancytokeratin antibodies. We identified at least one SLN in 47 of the 55 patients (85%) and examined a median of 26 lymph nodes per patient (range 10-59). By routine H&E staining, 14 of the 47 patients showed lymph node metastases (30%); the remaining 33 were classified as node-negative. In this group (33 patients), 1011 lymph nodes were analyzed by step sections and IHC: 14 of 70 SLNs. (20%) but only 37 of 941 non-SLNs (4%) had MM/ITC (p < 0.001). Furthermore, 13 of the 33 H&E-negative patients were found to have MM/ITC (39%). In 11 of the 13 patients, MM/ITC were identified in both SLNs and non-SLNs in 1 patient in the SLN only, and in 1 patient in a non-SLN only (sensitivity for the identification of MM/ITC: 92%; negative predictive value: 95%). The SLN biopsy is a valid tool to detect, as well as exclude, the presence of MM/ITC in colon cancer patients. Our results may be of prognostic relevance and influence patient stratification for adjuvant therapy trials.