BACKGROUND: Circulating autologous CD133+ stem cells have been differentiated into a number of cell types that have the potential for clinical use, including endothelial cells. These cells are infrequently found in peripheral blood specimens, and this limits their use in clinical applications. To address this problem, we have developed an extracorporeal cellular affinity (ECA) column that can recover CD133 expressing progenitor cells with high efficiency. METHODS AND RESULTS: In sheep model, 1800 mL of blood was passed through a Sepharose-based column with affinity for CD133. Unbound cells and plasma were returned to the animal. Our results show that this process has a minimal effect on the hematologic and physiologic parameters of the animal. This recovery approach generated over 600-fold more endothelial colony forming units than conventional buffy leukocytes isolated from a peripheral blood specimen. Ultimately, the increased cell recovery of the ECA column enables the generation of a cell biomass for therapeutic purposes in nearly a third of the time. CONCLUSIONS: This technology may facilitate the generation of large numbers of progenitor-derived cells for clinical therapies and reduce the time required to attain clinically relevant cell numbers while minimizing loss of other important cell types to the donor.