Hepatitis B virus X protein (HBX) has been implicated in the transactivation of diverse cellular genes and possibly also the pathogenesis of human hepatocellular carcinoma (HCC). We report the characterization of HEX variants from HBV-related human hepatocellular carcinoma (HCC). These HEX variants were integrated into the host chromosomes and also expressed in the HCC tissues. In addition, we report a novel in vitro HEX activity assay based on color changes that were indicative of the beta-galactosidase enzyme activity. Conducted in wheat germ lysates, the transactivating function of either wild type or mutant HEX protein was measured through their interaction with the Early Growth Response factor 1 (Egr-1) that controls the beta-galactosidase gene. Further analysis of these HEX deletion mutants using this assay may shed new insights on the significance of various mutations occurring in HCC-associated HEX.