Self-renewing, multipotent hematopoietic stem cells are highly enriched within the Lin- Thy1.1(lo)c-kit+ Sca-1+ subset of mouse bone marrow. However, heterogeneous expression within this population of certain cell surface markers raises the possibility that it may be further fractionated phenotypically and perhaps functionally. We previously identified alpha2-integrin (CD49b) as a surface marker with heterogeneous expression on Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ stem cells. To determine whether differences in alpha2 expression were indicative of differences in stem cell function, we purified alpha2- and alpha2hi stem cells by fluorescence-activated cell sorting and analyzed their function in long- and short-term hematopoietic reconstitution assays. Both alpha2- and alpha2hi cells could give rise to mature lymphoid and myeloid cells after transplantation into lethally irradiated congenic recipients. However, alpha2hi cells supported hematopoiesis for only a short time (<4 weeks), whereas alpha2- cells reproducibly yielded robust, long-term (>20 weeks) reconstitution, suggesting that alpha2- cells represent a more primitive population than do alpha2hi cells. Consistent with this idea, alpha2- Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ cells exhibited an approximately sixfold decreased frequency of spleen colony-forming units (day 12) versus alpha2hi cells. Furthermore, bone marrow cells isolated from animals transplanted >20 weeks previously with 20 alpha2- Lin(-/lo)Thy1.1(lo)c-kit+ Sca-1+ cells included both alpha2- and alpha2hi stem cells of donor origin, indicating that alpha2hi cells are likely lineal descendents of alpha2- cells. Interestingly, alpha2 integrin expression is significantly reduced on lineage-restricted oligopotent progenitors in the marrow, suggesting that high level expression of alpha2 selectively marks a subset of primitive hematopoietic cells which retains multilineage reconstitution potential but exhibits reduced self-renewal capacity.