Molecules bearing thymus leukemia (TL) alloantigen were isolated by immunoprecipitation from detergent-solubilized thymocyte lysates. Antisera used included monoclonal antibodies (anti-TL.m1, anti-TL.m2, anti-TL.m3), monospecific anti-TL.5 alloantisera and multispecific anti-TL.1,2,3,5 antiserum. Apparently, each of these reagents immunoprecipitates the same single 45,000 molecular weight Tla gene product as shown by identity on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), isoelectric focusing and by sequential precipitation studies. Allelic TL molecules, coded by the Tlaa and Tlad genes, were shown to be distinguishable by SDS-PAGE, and tryptic peptide mapping experiments. Both allelic TL molecules could be isolated from thymocytes of (Tlaa x Tlad)F1 mice. These results suggest that, at least for the Tlaa-Tlad allelic differences, the polymorphism and antigenicity of TL is determined by variation in amino acid composition.