Double immunofluorescence studies using both surface and cytoplasmic antigens were performed on cells of some human hematopoietic lines. We tested several permeabilization protocols in order to optimize, improve and simplify flow cytometric assay to detect the combinations of two markers present in one cell which could be regarded as leukemia-related markers. It was found, that buffered formaldehyde-acetone (BFA) fixation renders the cell membrane permeable without destroying surface antigens so that intracellular and cell surface markers could be measured simultaneously by flow cytometry. Cell lines used for the experiments reported here included MOLT4 T cell line, mature B cell lines DAUDI and U-266, and early B cell line REH-6. Results from our studies demonstrated, that in the absence of CD3 antigen on the surface membrane of viable MOLT4 blast cells, double labeling of fixed, permeabilized cells revealed 97% mCD7+, cCD3+ double positive cells. Two color staining with anti-CD19 and anti-CD22 monoclonal antibodies (MoAbs) in DAUDI cells showed, that larger part of cCD22+ cells expressed mCD19 antigen. CD22 antigen was absent on DAUDI cell membrane. Of great interest was the finding, that the marker detected by anti-CD19 MoAb which was absent on the membrane of U-266 cells was detected in their cytoplasm. Double staining of these cells revealed, that the number of mCD22+, cCD19+ double positive cells was 80%. Cytoplasmic CD22 antigen along with surface membrane CD19 was used to define early B cell line REH-6 as well. Our results demonstrate majority of double positive cells among tested population (mCD19+, cCD22+). To our knowledge the presence of cytoplasmic IgM detectable by flow cytometry in REH-6 cells, which could be so regarded as a precise and adequate counterpart to pre-B acute leukemia cell phenotype in children, is an original finding. Immunological typing plays an important part in the multiple marker analysis of hematopoietic malignancies. Through these surface and cytoplasmic marker combinations minor neoplastic cell populations could be detected. Human hematopoietic cell lines could serve as a reliable model system for monitoring minimal residual disease in acute leukemia patients.