We studied the mechanism of hematopoietic suppression in aplastic anemia by means of two-color flow microfluorometric analysis of lymphocyte subpopulations and correlated the results with the occurrence in vitro of hematopoietic suppression and interferon production. In 12 patients with aplastic anemia a striking increase was observed in a population of "activated" suppressor T lymphocytes, which were defined by binding of both anti-Leu-2 and anti-HLA-DR monoclonal antibodies (patients with aplastic anemia, 6.8±3.2 per cent [mean ±S.D.]; normal subjects, 1.7±1.3; patients given multiple transfusions, 2.5±1.7). Tac antigen expression, another surface marker of lymphocyte activation, was increased on suppressor lymphocytes in all five patients examined (patients with aplastic anemia, 31±17 per cent; normal subjects, 0.7±0.24; patients given multiple transfusions, 2.3±1.2). When Tac+ and Tac cells were separated in a cell sorter, only Tac+ cells produced interferon. When lymphocytes of patients with aplastic anemia were cocultured with normal bone marrow, only the Tac+ cell fraction showed hematopoietic suppressor activity. In one patient, in vitro elimination of suppressor lymphocytes by use of OKT8 antibody abolished spontaneous interferon production by bone-marrow cells. These results suggest that activated suppressor lymphocytes producing interferon have a role in the pathogenesis of bone-marrow failure, and indicate the usefulness of defined lymphokine and phenotypic markers in the study of aplastic anemia. (N Engl J Med 1985; 312: 257–65.)