Tumor rejection antigen (TRA) of an ultraviolet-light-induced murine skin tumor was solubilized, fractionated and partially characterized. Subcellular fractions were prepared by differential centrifugation of tumor cells that had been ruptured via nitrogen cavitation. Only the 110,000-g membrane fraction induced significant tumor protection, as determined by in vivo immunization and challenge assays. Extraction of the membrane fraction with 3 M KCI resulted in solubilization of material that could induce in vivo tumor-rejection immunity. Both the membrane fraction and soluble extract had a limited effective dose range. The KCI extract was separated on a Sepharose, CL-6B column in the presence of 6 M guanidine-HCI. Only one of five fraction pools (molecular weight range of 76,000-127,000 daltons) was immunogenic. It contained at least eight protein bands by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), but no lipid components. This immunogenic Sepharose fraction was chromatographed on a Sephadex G-150 column. Each of the four Sephadex fraction pools was immunogenic. One protein component was common to each of those fractions. It migrated as a single 76,000-dalton band on SDS-PAGE and contained [14C]-leucine and [3H]-glucosamine that had been incorporated during cell growth. These results suggest that the TRA of this tumor is a 76,000-dalton glycoprotein.