AIM: Contrast harmonic endosonography (CHEUS) is not widely available. This study assessed the utility of CHEUS using DEFINITY™, a second generation ultrasonic contrast agent, in the evaluation of suspected pancreatic and peri-ampullary malignancies. METHODS: Prospectively enrolled patients with suspected pancreatic and peri-ampullary malignancies underwent EUS followed by CHEUS. The incremental yield of CHEUS over EUS was analyzed. The gold standard for diagnosis of malignancy was positive cytology or histology; a negative diagnosis for malignancy was based on negative cytology or histology and benign clinical course. RESULTS: Twenty-nine patients were enrolled and underwent CHEUS. The final diagnoses were: pancreatic adenocarcinoma (16/29); metastases to pancreas (4/29); pancreatitis with inflammatory mass (4/29); normal pancreas with focal fat sparing (1/29); ampulla adenocarcinoma (2/29); serous cystic neoplasm (1/29); peri-pancreatic lymph node due to lymphoma (1/29). One bengin case of chronic pancreatitis had calcification casting artifacts that prevented accurate EUS examination and was excluded, leaving 28 cases for comparative analysis between EUS and CHEUS. CHEUS enhanced tumor margins. CHEUS detected vascular invasion missed by EUS in 2/16 patients with pancreatic adenocarcinoma. Masses appeared hypoechoic with EUS. With CHEUS malignant masses had an inhomogeneous hypoechoic pattern associated with abnormal vessels while lesions due to focal pancreatitis or fat sparing were characterized by diffuse enhancement (p<0.001). CONCLUSION: CHEUS improved the visualization of tumor margins and vascular invasion, and differentiated benign from malignant masses.