While adrenal gland histoplasmosis has been previously diagnosed by fine needle aspiration utilizing the percutaneous approach, EUS-FNA has not been employed in the diagnosis of this infection affecting both adrenal glands. We report a patient with massive bilateral adrenal enlargement due to histoplasmosis that was diagnosed by EUS-FNA. Trans-duodenal and transgastric fine needle aspiration biopsy of both adrenal glands was performed. Rapid onsite cytopathologic evaluation (ROSE) revealed epithelioid histiocytes, singly and in clusters consistent with granulomas. Apparent intracytoplasmic inclusions suggestive of organisms were visible. A Gomori Methenamine Silver stain (GMS) revealed abundant small intracellular budding yeasts, morphologically consistent with Histoplasma; the patient was admitted for amphotericin B intravenous infusion. His fever abated on the second day after amphotericin B was started. His urine Histoplasma antigen was positive. Fungal cultures from both adrenal EUS-FNA samples grew Histoplasma capsulatum. After a one week hospital stay, he was discharged home on itraconazole 200 mg po bid for one year. Four months after initiation of treatment, his urine Histoplasma antigen was undetectable. Nine months after initial diagnosis, the patient regained his energy level, and returned to work with complete resolution of his initial symptoms. This case highlights that EUS-FNA with ROSE can be a highly effective tool in the diagnosis of uncommon infections of the adrenal glands. © 2009 Wiley-Liss, Inc.