Background and Study Aims: Masses in the spleen can be sampled by endoscopic ultrasound-guided fine-needle aspiration (EUS-FNA) but the diagnosis of lymphoma using EUS-FNA and flow cytometry has not been reported. We report our experience with transgastric EUS-FNA and flow cytometry in the investigation of patients with suspected lymphoma of the spleen. Patients and Methods: All patients with splenic lesions that had been detected by computed tomography and who were referred for transgastric EUS-FNA over a 3-year period were enrolled in this study. The tissue obtained by EUS-FNA was evaluated by flow cytometry in all patients. Results: Six patients with splenic masses were enrolled (four men, two women; median age 58.5 years, range 41 - 82 years). The mean size of the short axis of the lesions was 37.8 mm (SD 23.76 mm) and the mean size of the long axis was 45.6 mm (SD 31.72 mm). EUS-FNA was performed successfully in all patients and the tissue obtained was evaluated by flow cytometry. Two patients were diagnosed with lymphoma; no pathology was identified in the other four patients. Lymphoma of the spleen appeared as sharply demarcated echo-poor lesions; benign lesions appeared echo-rich in comparison with the surrounding splenic tissue. The two patients who were diagnosed with lymphoma underwent chemotherapy. Of the four patients in whom no pathology was identified, one patient subsequently underwent splenectomy for evaluation of persistent abdominal pain and was diagnosed with lymphoma; the three other patients had true-negative disease on the evidence of long-term follow-up (mean 8 months; range 6 - 12 months). No complications related to the EUS-FNA procedure were encountered in any patient. Conclusions: EUS-FNA of spleen masses is a safe technique that aids in the diagnosis of lymphoma when used in conjunction with flow cytometry. © Georg Thieme Verlag KG Stuttgart.