The NOD/SCID xenotransplantation assay has been used extensively to develop ex vivo expansion cultures of human pluripotent hematopoietic stem cells, termed SCID populating cells (SRC). However, to definitively measure expansion of human SRC, quantitative analysis of SRC frequency before and after ex vivo culture is necessary. Here, CD34+CD38-LU1- cells, previously shown to be enriched for SRC, were seeded into wells at a dose near limiting dilution (1000 cells). The contents of each well was harvested and transplanted into individual mice at day 0 or following 10 days of ex vivo culture. As predicted by frequency analysis of mice transplanted with cells on day 0, 29% of the wells contained an SRC. However, after 10 days of ex vivo culture, the frequency of wells containing SRC had increased to 52%. Since the contents of individual wells were not combined during ex vivo culture, expansion of individual SRC in single wells seeded at day 0 may have contributed to the level of chimerism in individual mice transplanted, but could not have accounted for an increased frequency of wells containing populating cells originally seeded at day 0. We suggest that this increase in detectable SRC is due to populating cells that are undetectable at day 0 in NOD/SCID mice but require ex vivo culture prior to developing populating capacity. We have defined this unique human populating cell as a "Pre-SRC". Our study illustrates that caution must be exercised in the interpretation of ex vivo culture of human SRC, since augmentation of human populating cell number may not be a result of de novo isolated human SRC undergoing cell divisions, but may alternatively be due to detection of a Pre-SRC.