Replication-deficient adenoviral vectors (AdVec), which infect cycling and noncycling cells with high efficiency, low toxicity, and ease of delivery, provide ideal vehicles to study the expression of regulatory genes controlling different stages of hematopoiesis. To examine the infection efficiency of AdVec in hematopoietic precursor and progenitor cells, we used a replication-deficient adenovector expressing the humanized form of the cDNA for green fluorescent protein (AdGFP), permitting assessment of infection efficiency and kinetics of transgene expression in viable hematopoietic cells using flow cytometry and fluorescence microscopy. Flow-cytometric analysis of ex vivo expanded hematopoietic precursor cells infected with a multiplicity of infection (MOI) of 100 of AdGFP show that 78% of megakaryocytic (CD41a+ and CD42b+) cells, 82% of dendritic (CD1a+) cells, 41% of RBC precursors (glycophorin A+), and 32% of monocytic (CD14(+)) cells expressed GFP. Nineteen percent +/- 1% of freshly isolated CD34(+) cells from peripheral blood leukapheresis products infected under the same conditions expressed GFP. Morphologic evaluation of ex vivo expanded, AdGFP-infected CD34(+) cells showed normal maturation. The functional capacity of AdGFP-infected CD34(+) cells was analyzed by quantifying clonogeneic efficiency and proliferative capacity. Infection of CD34(+) progenitor cells with MOIs of 1 to 100 did not impair clonogeneic efficiency of CD34(+ )cells. However, MOI greater than 100 resulted in a significant inhibition of colony-forming unit-granulocyte/granulocyte-macrophage (CFU-G/GM) formation. In sequential dilution expansion over 3 weeks (Delta assay), the cytokine-driven proliferative potential of CD34(+) cells was not impaired following exposure to AdGFP at MOIs of 1 to 1,000. The GFP+ population expanded 10- to 15-fold at high MOIs (500 to 1,000), indicating multiple copies of the transgene in the initially infected CD34(+) cells, which were expressed in subsequent progenies. These data show that AdVec deliver transgenes with high efficiency and low toxicity to hematopoietic progenitor and precursor cells. Introduction of marker genes such as GFP into hematopoietic cells by AdVec will provide a valuable system for study of development, homing, and trafficking of hematopoietic precursor and progenitor cells in vitro and in vivo. Furthermore, these results provide insights into the design of gene therapy strategies for treatment of hematologic disorders by AdVec.