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Needle biopsy of splenic lesions using computed tomography (CT) or ultrasound (US) is difficult if the size of the lesion is small. It may be dangerous if the lesion is adjacent to the splenic hilum or located peripherally. We used endoscopic ultrasound–guided fine needle aspiration (EUS-FNA) to elucidate the tissue diagnosis of splenic abnormalities.EUS-FNA was performed in 12 patients when US- or CT-guided biopsy was inconclusive (n = 5), was not attempted because of small tumor size (0.9–1.4 cm; n = 4), or was considered dangerous (n = 3). A linear echo-endoscope and 22-gauge needles were used for cytology and bacteriology.The age of the patients was 19–68 yr (median 32 yr). Seven patients were male and five female. The size of the lesions was 0.8–4.2 cm (median 1.4 cm). Cytology was inadequate in one patient. Bacteriology was positive for Staphylococcus aureus and Serratia in one patient each, and cultures were positive for Mycobacterium tuberculosis in two patients. A positive diagnosis was made in 10 of 12 patients (83%). Final diagnoses were tuberculosis in two patients, Hodgkin's disease in two, sarcoidosis in two, abscesses in two, metastatic colon cancer in one, and infarction in one. Suspected recurrence of non-Hodgkin's lymphoma was not confirmed in one case. One patient experienced pain after puncture, but no hematoma was demonstrated on subsequent US examination.EUS-FNA cytodiagnosis in patients with unknown splenic lesions seems feasible, even in very small foci, when CT- or US-guided biopsy fails. Additional material for bacteriology may show benign treatable diseases such as abscesses or tuberculosis.