We have solubilized by limited papain digestion and partially purified the tumor rejection antigen, tumor-specific transplantation antigens (TSTA), from membranes of a simian virus 40-induced sarcoma. Uniform-sized materials with a molecular weight range of 50,000 have retained their tumor rejection activities through the purification procedures. The simian virus 40 TSTA have been separated from H-2 activity by affinity chromatography on concanavalin A columns and no evidence was found for H-2 antigens in the unbound fraction (I) of concanavalin A containing TSTA activity. A reduced yield from the crude soluble fraction was observed with Fraction I of concanavalin A material and this may indeed represent fragmentation of antigen during papain digestion. These results stand in contrast to purification of histocompatibility antigens (H-2alpha) using the same methods and techniques. Low concentrations of simian virus 40 TSTA crude soluble materials were nervertheless biologically active. A concentration as low as 4 mug protein provided 50% tumor rejection and 0.1 mug protein provided lymphocyte stimulation. Both assays reflected specificity of response.