The sera of mice surviving challenge with a Thy-1-negative variant of the thymoma RL male 1 contain antibodies which identify a new cell surface antigen (Ly RL male 1) present on cells of the T lineage. This antigen appears early in the development of the lineage and it can be detected on most thymocyte precursors. Its presence on prothymocytes can serve to distinguish these cells from their multipotential precursors. The antigen is present on many thymocytes, and dividing thymocytes are more susceptible to its cytotoxic activity than is the total population. Ly RL male 1-antigen-positive cells can be detected in peripheral lymphoid tissues by both functional assays and absorption. Treatment of peripheral lymphoid cells with the antiserum leads to significant reduction in MLR and helper activity but does not alter mitogen reactivity or lymphocytotoxicity. Animals with significant serum levels of anti-RL male 1 are deficient in their ability to produce IgG antibody to sheep erythrocytes.