The authors evaluated a new cell membrane permeabilization method for the flow cytometric detection of terminal deoxynucleotidyl transferase (TdT). In this method, gradient-separated leukocytes or unseparated blood or bone marrow cells were incubated in a commercially available diethylene glycol-based red blood cell lysing solution, which not only lyses red blood cells, but also permeabilizes leukocyte cell membranes; the light scattering properties of the cells are retained. The validity of the current method was demonstrated by the good concordance of the findings with previously published data as follows: (1) practically identical results were obtained when an established method for cell permeabilization was used in parallel on the same samples; (2) the proportion of TdT-positive cells in normal peripheral blood was negligible; (3) the proportion of TdT-positive cells in normal bone marrow averaged 1%, and a significant portion of TdT-positive cells in normal bone marrow expressed CD10 and CD34; and (4) TdT-positive cell populations were seen with the expected frequencies in various types of leukemia. This method for TdT flow cytometry provides significant advantages over previously used methods and is especially suitable for TdT detection in routine laboratories.