The expression of the glycosphingolipid antigens was investigated in human colorectal carcinomas and adjacent normal tissues obtained by surgical operation from six patients. The glycosphingolipids, including both non-acidic glycolipid and ganglioside, were extracted from the tissue specimens and separated by two-dimensional thin-layer chromatography of silica gel. A novel neutral glycolipid antigen migrating lower than Le(a) blood group antigen was detected in the cancerous tissues of all six patients, but not in normal regions in the same patients. The structure of the glycosphingolipid was partially characterized by gas chromatography and gas chromatography/mass spectrometry of alditol acetate and negative ion fast atom bombardment mass spectrometry (FAB/MS) of the intact glycolipid, and TLC-immunostaining with monoclonal anti-Le(a), -Le(b), -sialyl Le(a), and -Le(Y) antibody, respectively. The novel neutral glycosphingolipid in colorectal cancer was identified to be the III(4)IV(2)Fuc(2)Lc(4)Cer, Le(b) antigen of the human Lewis blood system. Direct FAB/MS showed the ceramide was composed mainly of 2-hydroxy tetradecanon and C18 phytosphingosine. In such individuals, the expression of Le(b) antigen in normal tissues is absent or minimal. Based on this study, we suggest that this Le(b) antigen may be a human distal colorectal cancer-associated antigen, which will be helpful for diagnosis and treatment of distal colorectal cancer.