AC133 is a molecule whose expression in the human hematopoietic system is restricted to a subset of CD34+ progenitor/stem cells with long-term repopulating ability. The antigenic features of these cells, like CD34+ cells, are described heterogeneous. The immunophenotypic profile of AC133+ cells, detected by means of dual-color flow cytometry, in bone marrow (BM), cytokine-mobilized peripheral blood (PB) and umbilical cord blood (UCB) was evaluated. The highest percentage of AC133+ cells was detected in mobilized PB despite not significantly different from that found in BM, but both are higher than that found in UCB. In addition, the highest percentage of CD34, HLA-DR and CD33 co-expressing AC133+ cells was observed in mobilized PB. Furthermore, UCB was found to be enriched in CD7+ and CD19+ cells and BM was found to be enriched in AC133+ cells co-expressing CDw90 and CD71. Our data confirm the immunophenotypic heterogeneity of cells expressing AC133 antigen, a promising new stem cell marker to be increasingly used as additional target for alternative identification and separation of early hematopoietic cells.