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AIM: To differentiate focal liver lesions based on enhancement patts using three-dimensional ultrasonography (3D US) with perflubutane-based contrast agent.METHODS: Two hundred and eighty two patients with focal liver lesions, including 168 hepatocellular carcinomas (HCCs), 63 metastases, 40 hemangiomas and 11 focal nodular hyperplasias (FNHs), were examined by 3D US with perflubutane-based contrast agent. Tomographic ultrasound images and sonographic angiograms were reconstructed. Among 282 lesions, enhancement patts of 163 lesions between January 2007 and October 2007 were analyzed retrospectively.Then from November 2007 to May 2008, comparedwith contrast-enhanced (CE) 2D US, CE 3D US wasperformed on 119 lesions for prospective differentialdiagnosis. Sensitivity, specificity, area under receiveroperating characteristic curve (A z) and inter-readeragreement were assessed.RESULTS: With the tridimensional view, dominant enhancementpatts were revealed as diffuse enhancement or peripheral ring-like enhancement, followed with washout change for HCCs or metastases, respectively, and peripheral nodular enhancement or diffuse enhancement with spoke-wheel arteries, followed by persistent enhancement for hemangiomas or FNHs, respectively. At CE 3D US, the prospective differentiation of lesions showed sensitivity 92% (mean for two readers), specificity 91% and A z value 0.95 for HCCs, 84%, 97%, and 0.95 for metastases, 91%, 98%, and 0.98 for hemangiomas and 80%, 99%, and 0.99 for FNHs, respectively, while good to excellent inter-reader agreement was achieved. No significant difference exists between prospective diagnosis accuracy at CE 3D US and that at CE 2D US. CONCLUSION: CE 3D US provides a spatial perspective for liver tumor enhancement, and could help in differentiating focal liver lesions.