AIM:To clarify which method has accuracy:2nd gen-eration contrast-enhanced ultrasound or biopsy of portal vein thrombus in the differential diagnosis of portal vein thrombosis.METHODS:One hundred and eighty-six patients with hepatocellular carcinoma and portal vein thrombosis underwent in blinded fashion a 2nd generation contrast-enhanced ultrasound and biopsy of portal vein thrombus;both results were examined on the basis of the follow-up of patients compared to reference-standard.RESULTS:One hundred and eight patients completed the study.Benign thrombosis on 2nd generation contrast-enhanced ultrasound was characterised by progressive hypoenhancing of the thrombus;in malignant portal vein thrombosis there was a precocious homo-geneous enhancement of the thrombus.On follow-up there were 50 of 108 patients with benign thrombosis:all were correctly diagnosed by both methods.There were 58 of 108 patients with malignant thrombosis:amongst these,52 were correctly diagnosed by both methods,the remainder did not present malignant cells on portal vein thrombus biopsy and showed on 2nd generation contrast-enhanced ultrasound an inho-mogeneous enhancement pattern.A new biopsy during the follow-up,guided to the area of thrombus that showed up on 2nd generation contrast-enhanced ultra-sound,demonstrated an enhancing pattern indicating malignant cells.CONCLUSION:In patients with hepatocellular carcinoma complicated by portal vein thrombosis,2nd generation contrast-enhanced ultrasound of portal vein thrombus is very useful in assessing the benign or malignant nature of the thrombus.Puncture biopsy of thrombus is usually accurate but presents some sampling errors,so,when pathological results are required,2nd generation contrast-enhanced ultrasound could guide the sampling needle to the correct area of the thrombus. AIM: To clarify which method has accuracy: 2nd gen-eration contrast-enhanced ultrasound or biopsy of portal vein thrombus in the differential diagnosis of portal vein thrombosis. METHODS: One hundred and eighty-six patients with hepatocellular carcinoma and portal vein thrombosis underwent in blinded fashion a 2nd generation contrast-enhanced ultrasound and biopsy of portal vein thrombus; both results were examined on the basis of the follow-up of patients compared to reference-standard .RESULTS: One hundred and eight patients completed the study.Benign thrombosis on 2nd generation contrast-enhanced ultrasound was characterized by progressive hypoenhancing of the thrombus; in malignant portal vein thrombosis there was a precocious homo-geneous enhancement of the thrombus. On follow-up there were 50 of 108 patients with benign thrombosis: all were correctly diagnosed by both methods. There were 58 of 108 patients with malignant thrombosis: among these, 52 were correctly diagnosed by both methods, t he remainder did not present malignant cells on portal vein thrombus biopsy and showed on 2nd generation contrast-enhanced ultrasound an in-mogeneous enhancement pattern. A new biopsy during the follow-up, guided to the area of ​​thrombus that showed up on 2nd generation contrast -enhanced ultra-sound, demonstrated an enhancing pattern indicating malignant cells. CONCLUSION: In patients with hepatocellular carcinoma complicated by portal vein thrombosis, 2nd generation contrast-enhanced ultrasound of portal vein thrombus is very useful in assessing the benign or malignant nature of the thrombus . Puncture biopsy of thrombus is usually accurate but presents some sampling errors, so, when pathological results are required, 2nd generation contrast-enhanced ultrasound could guide the sampling needle to the correct area of ​​the thrombus.