In this "sandwich"-type enzyme immunoassay of galactosyltransferase isoenzyme II (GT-II), we used the monoclonal antibody MAb 3872 previously established and characterized to be specific for GT-II. Plastic beads coated with MAb 3872 were incubated with serum and buffer, washed, then incubated with MAb 3872 conjugated with horseradish peroxidase and again washed. Peroxidase activity remaining on the beads was measured by color development with o-phenylenediamine. The assay standard curve was linear from 0 to 150 kU/L. Inter- and intra-assay CVs were less than 9% and less than 7%, respectively. Mean GT-II values for 370 normal controls were 6.8 (SD 3.4) kU/L with no significant sex-, smoking-, or blood-type-related differences. We also assayed serum from patients with 11 different cancers or with matched benign diseases. The diagnostic sensitivity of GT-II for liver and esophageal cancer was 0.91 and 0.72, respectively, apparently higher than for carcinoembryonic antigen (CEA) or alpha-fetoprotein. GT-II and CEA concentrations in sera were not correlated. Evidently this assay may be useful as a cancer diagnostic test to supplement existing serological methods.