Background: The EUS appearance of lymph nodes in patients undergoing evaluation for malignancy was studied. Methods: In 378 patients EUS characteristics were assessed: size of tumor, largest lymph node size, number of lymph nodes, distance of lymph nodes from the tumor, and on a 5-point visual analogue scale(1 = least malignant to 5 = most malignant), roundness, homogeneity, and echogenicity, A morphology score (sum of roundness, homogeneity, and echogenicity) was calculated. Findings were compared with fine needle aspiration or surgical histopathology in 238 patients. Results: When using multivariate analysis, only lymph node size (1.4 +/- 0.1 cm vs. 1.7 +/- 0.1 cm, p = 0.001), distance from tumor (3.1 +/- 0.4 cm vs. 1.6 +/- 0.4 cm, p = 0.013), and morphology score (9.9 +/- 0.3 vs. 11.9 +/- 0.3, p = 0.001) for benign versus malignant lymph nodes (mean +/- SEM) were significant. Lymph node size was significant for esophageal cancer (p = 0.006) and other mediastinal lymph nodes (p = 0.007) but not for pancreaticobiliary malignancies (p = 0.1) or celiac lymph nodes (p = 0.4). Distance from the tumor was significant for pancreaticobiliary (p = 0.01) but not esophageal cancers (p = 0.7). Morphology was significant for all sites. The presence of at least one lymph node of 1 cm or greater within 1 cm of the tumor and with a morphology score of 14 or greater had a positive predictive value of 81% (prevalence = 13%). A morphology score of 6 or less had a negative predictive value of 92% (prevalence = 12%). Conclusions: Patients with malignant adenopathy have lymph nodes that are larger, closer to the primary tumor, rounder, darker, and more homogeneous than those of patients without malignant adenopathy.