Human cord blood (CB)–derived CD133⁺ cells carry characteristics of primitive hematopoietic cells and proffer an alternative for CD34⁺ cells in hematopoietic stem cell (HSC) transplantation. To characterize the CD133⁺ cell population on a genetic level, a global expression analysis of CD133⁺ cells was performed using oligonucleotide microarrays. CD133⁺ cells were purified from four fresh CB units by immunomagnetic selection. All four CD133⁺ samples showed significant similarity in their gene expression pattern, whereas they differed clearly from the CD133⁺ control samples. In all, 690 transcripts were differentially expressed between CD133⁺ and CD133⁺ cells. Of these, 393 were increased and 297 were decreased in CD133⁺ cells. The highest overexpression was noted in genes associated with metabolism, cellular physiological processes, cell communication, and development. A set of 257 transcripts expressed solely in the CD133⁺ cell population was identified. Colony-forming unit (CFU) assay was used to detect the clonal progeny of precursors present in the studied cell populations. The results demonstrate that CD133⁺ cells express primitive markers and possess clonogenic progenitor capacity. This study provides a gene expression profile for human CD133⁺ cells. It presents a set of genes that may be used to unravel the properties of the CD133⁺ cell population, assumed to be highly enriched in HSCs.