The mAb A33 detects a membrane antigen that is expressed in normal human colonic and small bowel epithelium and > 95% of human colon cancers. It is absent from most other human tissues and tumor types. The murine A33 mAb has been shown to target colon cancer in clinical trials, and the therapeutic potential of a humanized antibody is currently being evaluated. Using detergent extracts of the human colon carcinoma cell lines LIM1215 and SW1222, in which the antigen is highly expressed, the molecule was purified, yielding a 43-kDa protein. The N-terminal sequence was determined and further internal peptide sequence obtained following enzymatic cleavage. Degenerate primers were used in PCRs to produce a probe to screen a LIM1215 cDNA library, yielding clones that enabled us to deduce the complete amino acid sequence of the A33 antigen and express the protein. The available data bases have been searched and reveal no overall sequence similarities with known proteins. Based on a hydrophilicity plot, the A33 protein has three distinct structural domains: an extracellular region of 213 amino acids (which, by sequence alignment of conserved residues, contains two putative immunoglobulin-like domains), a single hydrophobic transmembrane domain, and a highly polar intracellular tail containing four consecutive cysteine residues. These data indicate that the A33 antigen is a novel cell surface receptor or cell adhesion molecule in the immunoglobulin superfamily.