Circulating CD133+ stem cells from the peripheral blood have been shown to be able to differentiate into numerous cell lineages. However, adults have only a small number of these circulating stem cells. The aim of the present study was to assess a new isolation and enrichment technique for CD133+ stem cells from peripheral blood with the use of Percoll density gradients. Our results demonstrated the presence of two large mononuclear bands when whole blood was centrifuged with 48% and 50% Percoll concentrations. Flow cytometric analysis (FACS) revealed a major CD133+ enrichment at the 48% Percoll concentration in one of the two bands. Further culture of these cells resulted in the formation of multiple colony-forming units. Our results suggest an advantage from using a simple Percoll gradient for successful CD133+ cell recovery, which could aid in differentiation and transplantation protocols.