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Background and study aims This study was designed to evaluate the impact of additional tissue obtained with endoscopic ultrasound (EUS)-guided 25-gauge core biopsy needle (25G-PC) following an unsuccessful fine-needle biopsy (FNB) performed with larger-bore needles for the characterization of gastrointestinal subepithelial lesions (GI-SELs). Patients and methods We prospectively collected and retrospectively analyzed information in our database from January 2013 to June 2017 for all patients with GI-SELs who received a EUS-guided FNB (EUS-FNB) with 25G-PC during the same procedure after failure of biopsy performed with larger-bore needle. Diagnostic yield, diagnostic accuracy and procedural complications were evaluated. Results Sixteen patients were included in this study, 10 men and 6 women, median age 67.8 (range 43 to 76 years). Five patients were found to have a SEL localized in the distal duodenum, five in the gastric antrum, two in the gastric fundus and four in the gastric body. The mean size of the lesions was 20.5 mm (range 18 – 24 mm). EUS-FNB with 25G-PC enabled final diagnosis in nine patients (56.2 %). Regarding the subgroup of duodenal lesions, the procedure was successful in four of five (80 %). Final diagnoses with EUS-guided sampling were GIST (n = 6), leiomyoma (n = 2) and metastatic ovarian carcinoma (n = 1). No procedure-related complications were recorded. Conclusion In patients with small GI-SELs, additional tissue obtained with 25G-PC could represents a “rescue” strategy after an unsuccessful procedure with larger-bore needles, especially when lesions are localized in the distal duodenum.