Monoclonal IgG and IgM antibodies (mAbs) directed against serum galactosyltransferase (GalTase) activities were prepared and characterized for their relative specificity for GalTase isoenzymes I and II (GalTase I and GalTase II). After immunization of mice with purified GalTase, 7 of 1680 fusion products screened were positive for anti-GalTase activity in a solid-phase assay; of these 7, 2 were found to bind GalTase I in a somewhat selective manner while 1 (C6) was relatively specific for GalTase II. The Ka for anti-GalTase mAbs ranged from 2.7 X 10(7) to 1.1 X 10(8) M-1. Enzymatically active GalTase could be recovered from an affinity column of C6 coupled to Sepharose 4B following application of a cell extract from a human colon cancer cell line, confirming that the mAb is directed against GalTase. A sandwich RIA was developed to measure soluble GalTase II in serum by using a combination of two isoenzyme nonspecific mAbs (F5 and V10) coated on a solid-phase support followed by the addition of antigen and GalTase II-specific 125I-labeled C6. This assay was specific for GalTase II with a sensitivity of approximately 10 ng/ml. Evaluation of 240 sera demonstrated higher levels of GalTase II in patients with gastrointestinal cancer (48 ng/ml). However, in contrast to previous results with a radiochemical assay, some normal sera contained GalTase II (mean, 14 ng/ml) and some patients with liver disease had elevated levels (mean, 23 ng/ml). These studies demonstrate the production of moderate-affinity antibodies directed to serum GalTase isoenzymes and the development of an RIA useful in the study of GalTase.