We determined the surface phenotype of leukemic cells in adult T-cell leukemia (ATL) by immunofluorescence. Peripheral blood lymphocytes of 10 ATL patients were stained with monoclonal antibodies either directed to the surface antigens of all peripheral T cells (Leu-1), suppressor/cytotoxic (Leu-2a) T cells, or helper/inducer (Leu-3a) T cells. In addition, staining with the heterologous anti-glycolipid antiserum (anti-asialo GM 1) was also examined. Cytofluorographic analyses revealed the absence of anti-Leu-2a and anti-asialo GM 1-positive cell populations in all 10 cases of ATL studied. In contrast, the phenotypes of T-chronic lymphocyte leukemia cells were more variable from patient to patient. Morphological examination of fluorescence-activated cell sorter-separated Leu-1- or Leu-3a-reactive cells from ATL patients strongly suggested that the leukemic cells themselves expressed anti-Leu-1- and anti-Leu-3a-reactive antigens. From the above findings, we believe that these antibodies will be useful not only for the diagnosis of leukemia but also for the understanding of T-cell ontogeny and pathogenesis of leukemia in man.