Aim: Compared to standard two-dimensional (2D) endosonography, three-dimensional (3D) endosonography has been presented as a possible improvement regarding imaging of the gastrointestinal (G-I) tract and detection of metastatic lymph nodes. The aim of this study was to evaluate the efficacy of detecting periintestinal lymph nodes in surgical specimens using 3D endosonography. Patients and methods: Surgical specimens from 31 patients with malignant G-I tumours were investigated by 3-D endosonography and histology with focus on the presence of periintestinal lymph nodes and presence of metastasis. The specimens were scanned submerged into water. Position and size of the lymph nodes were mapped on a photo of the specimen both by the pathologist and the examiners. Results: Three-dimensional endosonography detected 48 out of 60 malignant lymph nodes (80.0%), and 110 out of 219 benign lymph nodes (50.2%). The positive predictive value for an endosonographic finding interpreted as a lymph node was 0.97. Conclusion: The detection rates for periintestinal lymph nodes were relatively high and seemed superior to the one usually assigned to 2D endosonography. Although distinguishing between metastatic and non-metastatic lymph nodes remains a problem, all patients with histologically confirmed metastasis to lymph nodes were detected by 3D endosonography, and the technique thus seems suitable for grouping of patients prior to surgery.