alpha-Fetoprotein (AFP) is a well-known tumor marker of hepatocellular carcinoma (HCC). Monoclonal antibodies against AFP possessing specific binding ability to HCC are potential candidates for immunoscintigraphy and immunotherapy. A new monoclonal antibody against AFP (0325-6-9) was isolated. Its specificity and targeting tumor ability were characterized by enzyme-linked immunosorbent assay (ELISA), cell immunostain and complement killing. These results suggest that 0325-6-9 is specific to hepatoma cells. The nucleotide sequences of variable regions of 0325-6-9 were determined by M13 dideoxynucleotide sequencing method. With the information of nucleotide sequence, this antibody then could be modified by recombinant technology for its usage in in vivo diagnosis and immunotherapy.