FACScan immunophenotyping of sorted human circulating CD133⁺ and CD133^- cells. Human circulating CD133⁺ cells obtained from the peripheral blood of healthy donors were characterized after FACSVantage sorting for the expression of adhesion molecules and chemokine receptors. The expression of the adhesion molecules was also evaluated in sorted CD133^- cells and after stimulation of sorted CD133⁺ cells with 5 ng/mL TNF-α. CD133⁺ cells (99% ± 1%) coexpressed CD44 and LFA-1. In particular, we observed 2 subpopulations of CD133 on the basis of LFA-1^dim and LFA-1^bright expression. In addition, 83% ± 2% of CD133⁺ cells expressed PSGL-1, 44% ± 3% expressed VLA-4, and 60% ± 5% expressed L-selectin. Analysis of chemokine receptors showed a very low expression of CXCR3 and CXCR4 (4% ± 3%), whereas 44% ± 5.4% of CD133⁺ cells were positive for CCR7. Less than 2% of total CD133⁺ cells expressed CCR3 (1.6% ± 1.3%) or CCR5 (1.7% ± 1.5%). We observed that sorted CD133^- cells expressed 98% ± 1% CD44, 87% ± 1% LFA-1, 63.5% ± 1% L-selectin, 53.5% ± 1% CCR7, 19% ± 1% CXCR3, 3.5% ± 1% CXCR4, 1.2% ± 1% CCR3, and 0.09% ± 1% CCR5. Very low expression of VLA-4 (0.6% ± 0.5%), CCR3 (1.2% ± 1%), and CCR5 (0.09% ± 1%) was observed. After cytokine stimulation of sorted CD133⁺ cells, the expression of L-selectin decreased from 60% to 4%. The other adhesion and chemokine molecules showed similar decreases.