A microplate leukocyte adherence inhibition (micro-LAI) assay was performed with peripheral blood mononuclear cells obtained from patients with hepatoma and control subjects (including healthy donors and patients with other diseases). Cell extracts of human hepatoma cells (HCC-M) and human hepatic cells (Chang liver cell) in tissue culture were prepared by sonication followed by centrifugation. The supernatants of these two cell lines were used as a specific antigen and a nonspecific antigen, respectively. It was found that monocytes were major indicator cells and that monocytes produced an LAI reaction in the absence of lymphocytes. Therefore, a repeated microplate monocyte adherence inhibition (MAI) assay was developed, in which the monocyte population of adherent cells is increased by removing nonadherent cells after an initial assay in fetal calf serum-containing medium without test antigens, and monocytes are counted selectively as peroxidase-positive cells in a subsequent second assay with test antigens. With regard to sensitivity and reproducibility, the repeated micro-MAI assay is superior to a micro-MAI assay in which the initial assay is omitted although monocytes are selectively counted. With this simple and sensitive technique a hepatoma-associated immune response to the extract of HCC-M was detected in 16 out of 22 patients (73%) with hepatoma, whereas the false-positive rate was 7% (3/41) in all control subjects.