Caspase-3 is a downstream effector cysteine protease in the apoptotic pathway. It is ubiquitously expressed in normal human tissues including the liver. Overexpression and loss of expression of caspase-3 has been reported in diverse human malignancies. However, expression of caspase-3 in hepatocellular carcinoma (HCC) has not been studied. Therefore, we studied its expression in four hepatoma cell lines and 22 HCCs by Western blot, and correlated the findings with in vitro caspase-3 activity and apoptosis. In addition, 47 surgically resected HCCs and 29 metastatic colorectal carcinomas were evaluated for caspase-3 expression by immunohistochemistry on formalin-fixed, paraffin-embedded tissue sections, and the staining intensity was correlated with the clinicopathological features. Caspase-3 overexpression was present in all four hepatoma cell lines, and 68% (15/22) of HCCs in comparison to the non-neoplastic liver parenchyma by Western blot, and in 52% (36/69) of HCCs by immunohistochemistry. Caspase-3 overexpression in HCCs by Western blot correlated with caspase-3 overexpression by immunohistochemistry (P=0.002), and in vitro caspase-3 activity (P=0.01). Caspase-3 overexpression in HCCs by immunohistochemistry was associated with serum alpha-fetoprotein (AFP) levels (P=0.01). In conclusion, caspase-3 is frequently overexpressed in HCCs and is associated with high serum levels of AFP.