B-cell precursor ALL (B-ALL) are generally believed to express genes, including those encoding surface antigens like CD19, CD22 and CD38, which correspond to those normally expressed by B-cell progenitors. Our previous efforts to immunophenotypically isolate normal HSC from B-ALL marrow for autologous transplantation demonstrated that patients’ putative normal CD34+ CD38− HSC populations contained leukemic cells, even though the B-ALL was positive for CD38 expression. These results raised the question of whether B-ALL stem cells, like normal HSC and ANLL stem cells, might be CD34+ CD38− . Therefore, gene expression was analyzed in rare CD34+ CD38− and more common CD34+ CD38+ B-ALL cells, and in normal CD34+ CD38− HSC and CD34+ CD38+ CD19+ B cell progenitors (BCP). RNA from stringently FACS isolated cells from marrow samples of >10 different B-ALL patients were amplified and hybridized to Affymetrix U133A microarrays. CD34+ CD38+ B-ALL cells expressed numerous genes expected of either B-ALL or normal BCP, including CD19, CD22, CD79a/b, Tdt, RAGs, Pax5 which were not expressed by normal CD34+ CD38− HSC. The expression pattern of the CD34+ CD38− B-ALL cells was different from that of the CD34+ CD38+ B-ALL cells, and from normal HSC or BCP. Compared to normal CD34+ CD38− HSC, the CD34+ CD38− B-ALL cells also expressed several B cell differentiation markers including CD19, CD22 and CD79, but underexpressed CD59, a modulator of responses to decay accelerating factor. There were also differences between the CD34+ CD38− B-ALL cells and the CD34+ CD38+ B-ALL cells. As predicted, CD34+ CD38− B-ALL cells underexpressed CD38, but unexpectedly lacked Tdt. The CD34+ CD38− B-ALL cells expressed multiple genes involved in cell cycle checkpoint control (cullin, MAD2, BUB3, MCMs, DDA3), survival and cellular/genomic maintenance (IAPs, translin), and immortalization (prohibitin), which were not expressed by the CD38+ B-ALL cells. FACS analyses of newly diagnosed patients with B-ALL demonstrated a rare population of CD34+ CD38− CD19+ leukemia cells, confirming that the CD19 mRNA found by array was translated. B-ALL consists of at least two subpopulations, one of which (CD34+ CD38− CD19+) has features that distinguish it both from normal HSC and the majority of B-ALL cells in the marrow. Besides their unique immunophenotype, the CD34+ CD38− CD19+ B-ALL cells express genes likely to be important for processes needed to maintain a malignant stem cell population. Potential implications of a phenotypically identifiable B-ALL stem cell include improved understanding of the biology of ALL and its therapy, the confounding results of MRD analyses, and the limitations of array analyses of unfrationated leukemic cells.