Galactosyltransferase (GT) (EC 2.4.1.38) was purified to homogeneity from human ovarian tumor effusion fluid and normal human serum by chromatography on alpha-lactalbumin and anti-human immunoglobulin affinity (to selectively absorb contaminating IgG) columns. Both preparations showed a single, broad band on sodium dodecyl sulfate-polyacrylamide gel electrophoresis centered at a molecular weight of 48,000, but nondenaturing polyacrylamide gel electrophoresis of GT isolated from tumor effusion fluid revealed the presence of a series of oligomeric proteins possessing GT activity, which were barely detectable in normal human serum. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of N-glycanase- and O-glycanase-treated GT revealed that each endoglycanase removed carbohydrate with an approximate molecular weight of 3,000, revealing the presence of both N-linked and O-linked oligosaccharide substitutions on GT. Purified GT (containing a mixture of GT isoenzymes) was used to immunize BALB/c mice for monoclonal antibody (MAb) preparation. Four of the MAb isolated reacted with GT. MAb 3872 (patent pending; an IgG1) was determined to be specific for a cancer-associated GT isoenzyme (GT-II) by immunostaining of Western blots and nondenaturing polyacrylamide gel electrophoresis of GT specifically eluted from a MAb 3872 affinity column. Two 125I-labeled cyanogen bromide peptides (Mr 8,400 and 7,400) prepared from 125I-GT were specifically bound and eluted from a MAb 3872 affinity column, demonstrating that the MAb 3872 GT-II-specific antigenic epitope resides on these peptides. MAb 3872 was immobilized on 1,1'-carbonyldiimidazole-activated trisacryl GF-2000 and used to specifically assay serum GT-II levels in 29 individual normal human serum samples and 77 serum samples from 38 patients with advanced ovarian tumors. The normal serum GT-II level was found to be 85.3 +/- 30.9 milliunits/ml, with a range of 17 to 160 milliunits/ml. Of the 38 tumor patients, 33 showed GT-II values in excess of 200 milliunits/ml, with a range of 216 to 8,469 milliunits/ml. Serial samples obtained from the ovarian tumor patients suggested that the serum GT-II level reflected the tumor burden of the patient.