The purpose of this study was to prepare and identify a novel monoclonal antibody (MAb) against human primary hepatocellular carcinoma (PHC) with high specificity and activity. The hepatocellular carcinoma cell line HepG2 was used to immunize BALB/c mice for preparing MAb with the classic hybridoma production technique. Tail vein injection immunization combined with intrasplenic injection was applied for improvement. Immunoperoxidase staining studies showed that the MAb was reactive to HepG2 and another hepatocellular carcinoma cell line, SMMC7721, 98.5% (67/68) specimens of hepatocellular carcinoma, but not to normal human liver tissues and tissues derived from the other malignant tumors, except one of the five specimens of cholangiocarcinoma with dubious staining. Laser confocal scanning microscope (LCSM) analysis indicated that the MAb reacted with the whole cell, including the membrane fractions and the cytoplasm. The hybridoma cell contained 103 +/- 5 chromosomes, and the MAb was identified as IgM subclass by ELISA. It was concluded that this combined immunization can effectively produce highly specific MAb against PHC, and this MAb may be of potential use as a targeting agent for radionuclide therapy and chemotherapy for hepatocellular carcinoma.