The monoclonal antibody (MAb) BD-5 reacts with an epitope (CAR-5) expressed in 83% of the gastric carcinomas and in 51% of the ductal pancreatic carcinomas. This MAb reacts also with epithelial cells of colorectal mucosa, but does not react at all with normal adult gastric mucosa or normal adult pancreas. We report the biochemical and immunochemical characterization of CAR-5-bearing molecule. The epitope was found to be carried on a mucin of more than 400 kDa with a density of 1.45 /ml, metabolicall labelled with 35S-sulfate, ity survived metaperiodate oxidation and alkalinization, while it was fully destroyed by pronase or papain. Trypsin, although cleaving the molecule, did not affect its antigenic activity. CAR-5 epitope is thus carried on the protein moiety of a sulfo-mucin. On the basis of its biochemical properties, the antigen was purified by a 3-step procedure, consisting of perchloric acid extraction, molecular sieving on Sepharose CL4B and affinity chromatography on wheat-germ agglutinin coupled to Sepharose 48. Cross-competition experiments, together with the chemical properties displayed by the different epitopes, clearly indicate that CAR-5 is different from all previously characterized carcinoma-associated determinants. Cross-DDIRMA experiments performed with different "catcher" and "tracer" antibody combinations showed that CAR-5 epitope may be expressed on the same mucin bearing CA 19-9, MOv2, DU-PAN-2, Lewis' and Lewisb epitopes. 3H-glucosamine, ! H mannose and r S-methionine. Antigenic-'To whom reprint requests should be sent, at Dept. of Biomedical Sciences and Human Oncology, C.so M.