A monoclonal antibody (designated L17F12) detects an antigen present on 95-100% of human peripheral T lymphocytes, the majority of thymocytes, and acute lymphocytic leukemia T cells but not B cells, B-cell lines, or monocytes. Examination of frozen tissue sections by the immunoperoxidase method revealed that the cells expressing this antigen were found predominantly in the medulla of thymus and in T-cell zones of lymph node and spleen. The antigen recognized by L17F12 was associated with a cell-surface glycoprotein of 67,000 daltons. L17F12 was used to isolate this molecule from human thymocytes, normal peripheral T cells, leukemic T cells, and T-cell lines. Expression of this antigen on normal T cells was not diminished by prolonged exposure in vitro to various T-cell stimuli. In the absence of complement, L17F12 bound to T cells without altering proliferative functions, thus enabling rapid purification of functionally intact T cells. In the presence of complement, L17F12 was cytolytic for T cells, providing the basis for depletion of T cells from heterogeneous populations. These data suggest that the monoclonal antibody L17F12 recognizes a specific T-cell differentiation protein. This antibody will be useful in studies of the human immune system.