Background: The diagnosis of intra-abdominal lesions of undetermined origin is often a challenge for endoscopists and radiologists. Objective: To evaluate the microvasculature of benign and malignant intra-abdominal lesions by contrast-enhanced harmonic EUS (CEH-EUS) and to investigate its usefulness for discriminating between malignant and benign lesions. Design: The vascularity of intra-abdominal lesions of undetermined origin was observed by using CEH-EUS. The lesions were classified according to their vascular patterns. The effectiveness of CEH-EUS in differentiating malignant from benign lesions was evaluated. Setting: Kinki University School of Medicine, Osaka, Japan. Patients: Forty-three patients, each with a lesion of undetermined origin, were evaluated prospectively by CEH-EUS between March 2007 and March 2009. Interventions: CEH-EUS was performed by using a prototype echoendoscope and the extended pure harmonic detection mode (a specific mode for contrast harmonic imaging). Main Outcome Measurements: The lesions were categorized by 2 physicians as having no, homogeneous, or heterogeneous enhancement. A consensus was reached for each case offline. How the benign and malignant groups differed in terms of their enhancement patterns was examined. Results: The κ coefficient of the interobserver agreement test was 0.953 (P < .001). Of the 27 malignant lesions, 26 (96.3%) exhibited heterogeneous enhancement. The 1 remaining malignant lesion (3.7%) showed homogeneous enhancement. Of the 16 benign lesions, none displayed heterogeneous enhancement, and 12 (75%) and 4 (25%) exhibited homogeneous and no enhancement, respectively. The malignant and benign lesion groups differed significantly in terms of homogeneous and heterogeneous enhancement (P < .001). The sensitivity, specificity, positive predictive value, negative predictive value, and accuracy with which CEH-EUS differentiated malignant from benign lesions were 96.3%, 100%, 100%, 94.1%, and 97.6%, respectively. Limitations: A single medical unit with a limited number of patients. Conclusions: CEH-EUS depicted the microvasculature of intra-abdominal lesions of undetermined origin very clearly and may be useful for characterizing such lesions. © 2010 American Society for Gastrointestinal Endoscopy.