Expression of decay-accelerating factor (DAF or CD55) and of CD59 during hematopoietic cell development in normal bone marrow and on peripheral blood leukocytes were characterized by three-color immunofluorescence experiments. With this technique cell subsets were identified by forward light scatter, orthogonal light scatter, and two cell-surface antigens. For each cell lineage, specific combinations of two monoclonal antibodies labeled with different fluorochromes were used. DAF or GD59 were then quantitated on the defined cell subsets from the fluorescence signal of the respective antibody conjugated with a third fluorochrome. Early uncommitted hematopoietic progenitor cells (CD34+, CD38⁺) all expressed both proteins homogeneously. Initial commitment to the eryth- roid (CD71⁺, CD45^dim), myeloid (CD33⁺), or B lymphocyte (CD10⁺) lineages was not associated with changes in DAF or CD59 levels. With erythroid development, i.e., after loss of CD45 and decrease of CD71, expression of both proteins decreased. With myeloid maturation, expression of GD59 remained constant and expression of DAF varied. During neutrophil maturation, DAF decreased initially and then reemeiged on maturing neutrophils concurrently with the appearance of GD16 (FcγRIII), whereas during monocyte maturation, DAF increased concurrently with up- regulation of CD14. With B cell development, expression of DAF increased concurrently with down-regulation of GD10 and up-regulation of CD20, whereas expression of CD59 diminished slightly late in B cell maturation. Analysis of peripheral blood elements showed that monocytes, neutrophils, and B lymphocytes expressed both proteins homogeneously, but that in contrast to other cell subsets, which all expressed CD59, only a subset of (CD3⁺) T lymphocytes and (GDI 6⁺) Natural killer cells expressed DAF. The absence of DAF was not related to CD4 or CD8 expression or to the presence of activation markers (CD25⁺, CD38⁺), memory cell markers (CD58⁺, CD45RO⁺), or virgin T cell markers (CD45RA⁺), but was correlated with expression of CDllb (CR3) and CDllc (gpl50/95). Although CD21” (CR2) and CD35* (CR1) cells all expressed DAF, CDlla (LFA-1) levels correlated inversely with those of DAF. Although the presence of CD55 and CD59 on early progenitor cells and throughout hematopoietic cell development is consistent with the requirements for both proteins in protection of host cells from complement-mediated injury, the physiological relevance of the unique patterns of variation for each cell lineage is unclear. Nevertheless, the availability of a detailed DAF and CD59 expression map in normal marrow will facilitate analyses of alterations during hematopoietic development that may occur in hematological disorders including paroxysmal nocturnal hemoglobinuria (PNH).