The carbohydrate moieties of glycoconjugates are thought to represent phenotypic variations in cancer cells most characteristically. In order to raise monoclonal antibodies recognizing these carbohydrate structures, Balb/c mice were immunized with human colonic cancer cell lines (LS 180 and SW 1116), and carbohydrate-directed antibodies were assayed using plastic plates coated with glycopeptides derived from cell surface glycoproteins of the cancer cells used for the immunization. Nine hybridoma clones have so far been established which were classified into four types based on their binding specificity: those binding only to mucin-type carbohydrates, those binding to all types of carbohydrates, i.e., mucin-type, serum-type carbohydrates and glycolipids, those binding to mucin-type carbohydrates and glycolipids, and those binding to serum-type carbohydrates and glycolipids. Another type of classification was also possible based on the involvement of sialic acid in the epitope: those in which removal of sialic acid abolishes the antigenicity, those in which removal of sialic acid enhances the antigenicity, and those in which sialic acid does not affect the antigenicity. The epitopic carbohydrate was identified as NeuAc alpha 2----6GalNAc for one of the antibodies, MLS 102, belonging to category. This is a heterophile antibody reacting strongly with ovine submaxillary mucin having a polyvalent antigenic structure. It was possible to stain colonic cancer cells with the antibodies differentially. Using two types of monoclonal antibodies, it was also possible to stain differentially clones constituting the cell line LS 180. The occurrence of at least four clones could be demonstrated. Such cell-staining capacities of the carbohydrate directed antibodies imply the importance of glycoconjugate carbohydrates as cancer cell phenotypes.