By immunization of rabbits with rat thymus cells a rabbit anti-rat thymocyte serum (ARTS) was obtained which reacted with thymic and peripheral lymphocytes, as shown by both specific immunofluorescence and cytotoxicity testing. Upon proper absorption or dilution this serum was rendered specific for rat thymocytes but cross-reacted with mouse thymocytes. The antigen which it defined was present in 100% of thymocytes in adult rats of all strains tested and in less than 7% of lymphocytes in adult spleen and lymph nodes. It was represented on three-quarters of the cells in both the thymus and spleen of the fetal rat but approached the adult distribution before the age of 4 weeks. ARTS also contained antibodies reactive with a second antigen present to an equal extent on both thymocytes and peripheral lymphocytes. Both antigens appeared to be distinct from that defined by the natural antibody in normal rabbit serum. It was suggested that the immunosuppressive effect of long term treatment with anti-thymocyte sera may depend in part on destruction of newly generated cells entering the peripheral pool from the thymus and bearing the thymus-specific antigen.