The objective was to assess EUS-FNA for diagnosing intramural upper GI tract lesions. The subjects were 50 patients (21M/29F) with upper GI submucosal lesions who underwent EUS-FNA at a referral center for GI system over a 12-month period. All cases were followed for 1 year after initial EUS-FNA. Cytologic diagnoses were categorized as benign, malignant, suspicious for malignancy, mesenchymal tumor, endocrine tumor, or nondiagnostic. All tumors were assessed for various cytomorphologic features. The accuracy of the initial FNA diagnoses was evaluated for each patient who also underwent subsequent histopathological examination of a core biopsy and/or surgical biopsy/resection material of the same lesion. According to the site of the lesions; while 84% of all esophageal lesions were diagnosed as mesenchymal; 67% of all gastric lesions were mesenchymal. The sole lesion was nonmesenchymal (benign cyst) in duodenum. The sensitivity, specificity, positive and negative predictive values, and accuracy of EUS-FNA for diagnosing submucosal mesenchymal tumors of the upper GI tract were 82.9, 73.3, 87.9, 64.7, and 80%, respectively. The corresponding values for nonmesenchymal lesions were 100, 85.7, 80, 100, and 90.9%. Our experience confirms that EUS-FNA is an extremely valuable tool for diagnosing submucosal lesions of the upper GI, and is particularly useful in cases where endoscopic forceps biopsy does not lead to diagnosis. Optimal results can be yielded by a close working relationship between the gastroenterologist and pathologist.