Autophagy is frequently activated in tumor cells treated with chemotherapy or irradiation; thus, regulation of autophagy represents a promising target for cancer therapy. However, the expression levels of autophagy proteins, Beclin1 and LC3, and their localization patterns in relation to mTOR pathway markers in patients with hepatocellular carcinoma has not been elucidated. Eighty-two tissue specimens (69 paraffin-embedded tissues and 13 frozen tissues) were obtained from patients with hepatectomy and expression levels of Beclin1, LC3, mTOR, and phospho-mTOR were analyzed by immunohistochemistry and western blots. Immunohistochemical analysis of 26 dysplastic nodules, 39 hepatocellular carcinomas, and 4 metastatic adenocarcinomas revealed a grade-dependent increase of immunostaining between grade II and III hepatocellular carcinoma for LC3 (P=0.02), Beclin1 (P=0.06), and mTOR (P < 0.01), and between grade I and II hepatocellular carcinoma for phospho-mTOR (P=0.05). A high proportion of positive immunostaining of autophagy and mTOR pathway markers was found in 18 grade III/IV hepatocellular carcinoma tissues (44-100%) and in 4 metastatic adenocarcinoma tissues (75-100%). Examining immunohistochemical staining scores revealed a strong correlation between mTOR and Beclin1 (R(2)=0.74) and intermediate correlations between LC3 and Beclin1 (R(2)=0.54) as well as between LC3 and phospho-mTOR (R(2)=0.56). Western blotting of 13 frozen hepatocellular carcinoma tissue specimens revealed a similar overall increase of LC3, Beclin1, and mTOR. This study demonstrated that Beclin1 and LC3 expression levels are increased in high-grade hepatocellular carcinoma and metastatic adenocarcinoma. Moreover, LC3 and phospho-mTOR showed different localization patterns, and increased autophagosome formation was observed in the tumor cells with LC3 positivity. Thus, autophagy is frequently activated in high-grade hepatocellular carcinoma, and regulation of the autophagy process may have diagnostic and therapeutic potential.