Summary A lymphocytopoietic fraction of calf thymic tissue, thymosin, has been utilized to prepare an antiserum in rabbits. The antiserum contained antibodies to at least two of the several components of the thymosin fraction. One of these precipitable antibodies was shown to be specific for a constituent of the thymosin preparation; the other was directed to bovine serum albumin. The antithymosin serum, after removal of antibody to BSA, was markedly cytotoxic to thymus cells but not to lymph node or spleen cells from calves, mice, and rabbits. The antithymosin serum agglutinated thymus cells of calves and cross-reacted with thymus cells of mice and rabbits. These activities of the antiserum to thymosin were not due to a Forssman antigen. The results support the concept of an antigenic difference between thymocytes and lymph node cells and provide evidence for the presence of a cellfree, soluble thymic antigen in thymosin.