We are investigating the antigenic changes on the cell surface of human hepatocytes that distinguish the normal from the transformed phenotype using monoclonal antibodies. In vivo expression of antigens has been directly assessed by in situ radioimmunohistology. This technique allows one to determine the distribution and density of antigen expression at the individual cellular level on fresh hepatoma and adjacent uninvolved liver tissue. We have found two antigens recognized by monoclonal antibodies XF-8 and AF-20 that are uniformly present on 15/15 hepatocellular carcinomas tested thus far. Most if not all tumor cells highly express these antigens. Such antigens were not evident on adjacent normal liver and the XF-8 epitope was not found on other normal human tissues. AF-20 antigen distribution revealed low-level expression on a subpopulation of cells in the zona glomerulosa of the adrenal gland and on crypt cells of the small intestinal tract. We have studied the capability of radiolabeled XF-8 and AF-20 monoclonal antibodies when administered either alone or in combination to localize a hepatitis B virus-related hepatocellular carcinoma cell line (FOCUS) grown as subcutaneous tumors in nude mice. Biodistribution experiments demonstrated an excellent localization to tumor of 15 to 22% of the injected dose of 125I-labeled antibodies. Indeed, it was possible to enhance the delivery of 125I to the tumor cell surface by the use of XF-8 and AF-20 in combination. Nuclear imaging studies showed sharp visualization of tumor and demonstrate that these monoclonal antibodies have sufficient specificity and sensitivity to be strongly considered as immunotargeting agents.