A subpopulation of normal thymocytes has been demonstrated which expresses a differentiated Lyt antigen cell surface phenotype similar to that of functional peripheral T cells. Cells with the phenotype Lyt 1 ion, 23- represent about 10% of normal thymocytes and are increased in vivo cortisone treatment to approximately 50%. Thymocytes can be separated by sedimentation velocity, density in BSA gradients, and peanut lectin agglutination. These separations can enrich for thymocytes of different Lyt phenotypes. A light density, large lymphocyte subpopulation can be obtained which expresses more Lyt 1 per cell than the majority of thymocytes and which is Lyt 23-. The majority of these cells do not appear to be agglutinated by peanut lectin and correspond to the cells obtained after in vivo cortisone treatment.