Side-population (SP) cells are a recently described rare cell population detected in selected tissues of various mammalian species, but not yet described in the peripheral circulation. In the present study, we have identified for the first time SP cells in lineage-negative adult human blood and have provided an extensive functional and immunophenotypic characterization of these cells. Adult peripheral blood was depleted of mature leukocyte cell types by density gradient and immunomagnetic separation. The SP cell population was identified by its characteristic Hoechst 33342 profile. Immunophenotypic analysis revealed that blood SP cells expressed high levels of CD45, CD59, CD43, CD49d, CD31, and integrin markers and lacked CD34. Highly purified SP cells were put into cobblestone area-forming cell (CAFC), long-term culture-initiating cell (LTC-IC), and liquid cell culture assays; repopulating assays were performed utilizing nonobese diabetic/severe combined immunodeficient mice. Circulating SP cells were shown to exhibit verapamil sensitivity and a low growth rate. LTC-IC, CAFC, and engraftment assays indicated that circulating SP cells had lost the multipotentiality described in murine bone marrow SP cells. However, outgrowth of mature cell types from liquid cell culture suggests the presence of common lymphoid (T and natural killer) and dendritic cell precursor(s) within circulating SP cell populations. The absence of SP cell growth in the LTC-IC, CAFC, and repopulating assays might be intrinsic to the tissue source (marrow versus blood) or species (mouse versus human) tested. Thus, human blood SP cells, although rare, may serve as a source of selected leukocyte progenitor cells. The immunophenotype of circulating SP cells may provide clues to their seeding and homing capacity.