Two monoclonal antibodies (KH-1 and KH-2) against a transplanted fibrosarcoma (KMT-17) in WKA rats were produced by fusing a mouse myeloma (P3-X63-Ag8.653) with spleen cells from syngeneic rats hyperimmunized with KMT-17. Both antibodies showed complement-dependent cytotoxicity against KMT-17. By absorption of cytotoxicity, KH-1 reacted with homologous tumor, other syngeneic fibrosarcomas (KMT-80 and KMT-75), and lung and kidney from normal rats. However, KH-2 reacted with many kinds of tumors and various normal tissues. Antigen specificity was tested by complement fixation and/or solidphase radioimmunoassay using glycolipids isolated from KMT-17 cells and authentic glycolipids. KH-1 reacted with globotriglycosyl ceramide which was not detected on KMT-17 cells and in cross-reacted weakly with IV3-α-galactosyl-lactoneotetraglycosyl ceramide, one of the major glycolipids of KMT-17. The immune reaction was inhibited by α-methyl-galactose. KH-2 reacted with lactosyl ceramide and lactoneotetraglycosyl ceramide. The reaction was more potently inhibited by lactose than by β-methyl-galactose. Antibodies with similar specificity to either KH-1 or KH-2 were elevated in syngeneic rat sera after serial immunization with viable KMT-17 cells.