Background: The ability to obtain tissue samples for histological examination during EUS has theoretical advantages over cytology alone. Objective: To prospectively evaluate the feasibility and yield of EUS-guided fine-needle tissue acquisition (EUS-FNTA) with a large-gauge needle in patients in whom we expected histology to be more useful than cytology to reach a definitive diagnosis. Design: Prospective cohort study. Setting: Tertiary care academic medical center. Patients: Consecutive patients with subepithelial lesions, esophagogastric wall thickening, mediastinal and abdominal masses/lymphadenopathy of unknown origin, or pancreatic lesions after nondiagnostic FNA. Interventions: EUS-FNTA with a 19-gauge needle. Main Outcome Measurements: Feasibility and yield of EUS-FNTA. Results: A total of 120 patients with a mean age of 61 +/- 14.6 years and mean lesion size of 38 +/- 25 mm (range 8-140 mm) were enrolled. FNTA was successfully performed in all but 1 patient (98.9%), and adequate samples for histological examination were obtained in 116 of the 119 patients (97.5%) in whom EUS-FNTA was technically successful. A mean of 2.8 +/- 0.8 passes per patient were performed. At the time of current follow-up, a definitive diagnosis was available in 117 of the 120 patients (97.5%), with only 8 false-negative results. The sensitivity, specificity, positive and negative predictive values, and diagnostic accuracy of EUS-FNTA in the 117 patients with a definitive diagnosis were 91.8%, 100%, 100%, 71.4%, and 93.2%, respectively. Limitations: Single-center study with limited power. Conclusions: EUS-FNTA by using a large-gauge needle has a high yield and promising diagnostic accuracy and could be used when histology may be more useful than cytology to reach a definitive diagnosis. (Gastrointest Endosc 2011;74:504-10.)