Objective: Endoscopic retrograde cholangiopancreaticography (ERCP)-guided brushing has been the standard of practice for surveillance and detection of carcinoma in the biliary tree. Few studies have evaluated the role of endoscopic ultrasound-guided fine needle aspiration (EUS-FNA) in diagnosing clinically suspected cholangiocarcinoma. The role of this method in diagnosing clinically suspected gallbladder malignancies has not been extensively evaluated in the USA. This study investigates the role of EUS-FNA in the diagnosis of clinically suspected biliary tree and gallbladder malignancies in a large patient series. Methods: EUS-FNAs were obtained from 46 bile duct and seven gallbladder lesions. On-site rapid interpretation was provided using air-dried Diff Quik stained smears. In addition, alcohol fixed Papanicoloau stained smears and Thin Prep preparations (q2Cytye Corp., Marlborough, MA, USA) were evaluated before providing a final cytological diagnosis. Tissue biopsies and/or clinical follow-up were used as the standards to determine operating characteristics for EUS-FNA. Results: The mean ages for bile duct and gallbladder lesions were 66 years (range: 37-84 years), and 69 years (range 49-86 years), respectively. All cases diagnosed as suspicious/malignant on preliminary evaluation were confirmed on final cytological interpretation (27/27). The operating characteristics show that EUS-FNA is highly specific (100%) with sensitivity rates of 87% and 80% from clinically suspected malignancies of biliary tract and gallbladder, respectively. Sampling error in three cases and associated acute inflammation in two cases resulted in false-negative diagnoses. Conclusions: EUS-FNA of biliary tree and gallbladder carcinoma is highly specific and should be considered for evaluation of clinically suspicious lesions. Marked inflammation may result in false-negative diagnoses. © 2006 Blackwell Publishing Ltd.