Monoclonal antibody (MAb) D612 recognizes an antigen expressed on the cell surface of normal and malignant gastrointestinal epithelium. It is a murine lgG2a/κ which has been previously shown to mediate killing of human colon carcinoma cells using human effector cells (which could be enhanced in the presence of interleukin-2). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analyses of MAb D612 immunoprecipitates of extracts of l-[35S]methionine-, l-[3H]leucine-, and d-[3H]glucosamine-labeled human colon carcinoma cells showed that the D612 antigen is a Mr 48,000 glycoprotein. Similar estimates of molecular mass were obtained from SDS-PAGE analyses of MAb D612 immunoprecipitates of radioiodinated extracts of surgically resected colon carcinoma and adjacent normal colonic mucosa. D612 antigen was not detectable in immunoprecipitates of supernatant media from radiolabeled cell cultures, suggesting that the antigen is not readily shed from the surface of cultured cells. The D612 antigen was shown to be clearly distinct from previously described gastrointestinal carcinoma-associated glycoproteins: the D612 antigen shows a migration pattern of SDS-PAGE distinct from those of the antigens recognized by MAbs KS1/4 and GA733, and reciprocal immunodepletion analyses of d-[3H]glucosamine-labeled colon carcinoma cells utilizing MAbs D612 and GA733 revealed no cross-reactivity between these antibodies. Similarly, competitive binding studies between MAbs 17-1A and KS1/4 and MAb D612 revealed no similarity between the epitopes recognized by MAb D612 and MAbs 17-1A and KS1/4. MAbs D612 and 17-1A were also titered in immunoperoxidase staining assays on serial frozen sections of normal and malignant colon. MAb D612 showed a higher titer of immunostaining reactivity with both normal and malignant colon than did MAb 17-1A. MAb D612 showed roughly equivalent immunostaining titers against normal and malignant colon; whereas MAb 17-1A showed a higher titer of immunostaining reactivity against the normal colon tissue than against the malignant colon. Flow cytometric analysis of phosphatidylinositol-specific phospholipase C-treated colon carcinoma cells revealed no loss of D612 antigen from the cell surface, suggesting that the mechanism of attachment of the D612 antigen to the cell surface does not involve linkage to a phosphatidylinositol glycan. Radioiodination of the D612 antigen in a plasma membrane-enriched cell fraction by the photoactivatable carbene-generating reagent, 3-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, suggests that the D612 antigen polypeptide penetrates the lipid bilayer of the plasma membrane. It has been determined by Scatchard analysis that the number of binding sites for MAb D612 on the LS-174T human colorectal carcinoma cell line is 4.8 × 105. MAb D612 was found to have a KA of approximately 1.3 × 109 m-1.