The molecular nature of a tumor-specific transplantation antigen (TSTA) of a chemically induced BALB/c mouse colon tumor C-C26 was investigated. The antigen was noncytolytically extracted by 2.5% n-butanol treatment of the cells. Crude butanol extract from C-C26, but not from colon tumor C-C51 and fibrosarcoma Meth-A of BALB/c mice could provide protection against the challenged C-C26 tumor in the transplantation experiment. Crude butanol extract from another syngeneic colon tumor C-C36 also induced a cross-protection against the challenged C-C26 tumor. C-C26 crude butanol extract was characterized by biochemical procedures including the Sephadex G200 column, lens culinaris affinity column, and anion-exchange Mono Q fast protein liquid chromatography column, and by the enzyme digestion study of the antigens and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The data indicated that C-C26 TSTA was eluted into fractions containing molecules of approximately Mr 200,000 on Sephadex G200 column chromatography. This antigen was also found in unbound fractions on a lens culinaris affinity column. The antigen was further separated into the fraction that was eluted with 0.4 M NaCl in an ionic strength on Mono Q fast protein liquid chromatography. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction showed the molecule with a molecular weight of 30,000. The enzyme digestion study indicated that the immunogenicity of the antigen was inactivated by papain but probably not by neuraminidase treatment. These data suggest that the immunogenic moiety of C-C26 TSTA molecules is located in the peptide portions rather than in sialic acid residues or carbohydrate portions. Furthermore, there are several similarities of the molecular characteristics between C-C26 TSTA and previously reported C-C36 TSTA, such as the amenability to n-butanol extraction. Lens culinaris lectin inaffinity, and ionic strength.