AIM: The purpose of this study is to evaluate the location of involved mesorectal and extramesorectal lymph nodes as depicted on preoperative MRI. Preoperative availability of this information might be useful for the surgeon as well as the radiation therapist and medical oncologist for optimal treatment strategy: type and extent of neoadjuvant treatment as well as extent of surgical resection. METHODS: Forty-one patients with biopsy-proven rectal cancer were included. All patients underwent preoperative MRI using USPIO (lymph node specific contrast agent). Location of all mesorectal and extramesorectal nodes visible on MRI was recorded, as well as USPIO prediction on nodal status. Lesion-by-lesion analysis using histology after surgery was performed for patients who did not receive long course chemoradiation therapy. RESULTS: There were 438 nodes visible, 94 of which were malignant. Most nodes are located in the laterodorsal part of the mesorectum, with no difference in distribution between positive and negative nodes. In relation to height of tumor, the majority of positive nodes are located at tumor height or above. There were significantly more negative nodes (9.6%) located below tumor height as compared to positive nodes (2.1%). There were 40 extramesorectal nodes, in 16 patients, 5 of which were positive in 4 patients. All patients had distal rectal cancer. CONCLUSION: In conclusion, positive mesorectal nodes are located in the laterodorsal part of the mesorectum, at tumor height or above. Positive nodes distal to the tumor are rare, and occur in patients with more proximal nodal metastases. Positive extramesorectal nodes mainly occur in patients with distal rectal cancer with nodal metastases in the mesorectum.