Agrin is a recently identified proteoglycan component of vascular and bile duct basement membranes in the liver. The selective deposition of agrin in hepatocellular carcinoma (HCC) microvessels versus sinusoidal walls prompted us to investigate the utility of agrin immunohistochemistry (IHC) in detecting malignant hepatocellular lesions. We focused on the differential diagnostic problems often presented by hepatocellular adenomas (HCAs) and dysplastic nodules. IHC for agrin was performed on 138 formalin-fixed, paraffin-embedded surgical specimens from 93 patients, including cirrhotic liver tissues (25), focal nodular hyperplasia (10), large regenerative nodules (8), low-grade (23) and high-grade (7) dysplastic nodules, small HCC (8), HCC (27), and HCA (30). Agrin immunostaining was compared with that of CD34 and, in selected cases, to glypican-3. The combination of agrin and CD34 sensitively (0.94) and specifically (0.93) identified lesions judged previously as malignant by histology. The majority of benign lesions were clearly agrin-negative, whereas the strength and extent of agrin IHC faithfully reflected dysplasia in "atypical" HCAs and in high-grade dysplastic nodules. Malignant lesions were uniformly positive. In conclusion, as agrin is highly selective for tumor blood vessels, IHC for agrin facilitates the discrimination of benign and malignant hepatocellular lesions. Moreover, whereas glypican-3 in some HCCs may appear in few scattered cells only, agrin is diffusely deposited in virtually all malignant lesions, which may prove advantageous in the evaluation of small specimens such as core biopsies.