BACKGROUND In 1996, Goodell et al. first described a rare subpopulation of bone marrow stem cells termed the Side Population (SP). SP cells are known to be CD34 negative and to have a high repopulating capability. The SP was identified by ultraviolet excitation based on the efflux of the DNA binding dye, Hoechst 33342 (Ho342). ABCG2, a halftransporter that belongs to the ATP binding cassette transporter superfamily, is the major contributor to the SP phenotype by actively pumping Ho432 selectively from stem cells. To date, very little is known about the identification of the SP in peripheral blood samples, and about its peripheral circulation, enrichment or isolation to evaluate its therapeutic potential. Due to the SP potential role in tissue regeneration, we studied the numbers of the SP in bone marrow and peripheral blood samples in regard to count accuracy and reproducibility. METHODS Bone marrow (BM) and apheresis (AP) specimens were obtained from healthy donors and patients undergoing stem cell transplantation. Bone marrow samples were obtained by aspiration. Peripheral blood cells after granulocyte colony stimulating factor (G-CSF) mobilization with or without chemotherapy, were obtained by apheresis. All samples were prepared for identification of SP cells by flow cytometry. RESULTS SP cells were detected in only 19 of 111 apheretic products, with relative frequency ranging from 0.01 to 4.75% of cells by the Ho342 exclusion method and flow cytometry analysis. Cell preparations used for these measurements consisted of 5 x 10(6) cells. However, no SP cells were detected when aliquots from the same positive specimens, consisting of previously stained 55 x 10(6) cells and fractionated into independent aliquots with 5 x 10(6) cells were used. CONCLUSIONS In this study, we show that there is great variability in SP cell numbers when aliquots obtained either from leukapheresis or bone marrow products represent about 1% of the total product volume. In contrast, when aliquots represented about 12% of the total product volume SP cells measurements were consistent. The high cell number of some specimens can be a limitation for the accurate identification and isolation of the SP compartment. Aliquots containing a minimum of 55 × 10(6) cells should be used for statistical significance.