Remarkable progress has been achieved in the characterization and isolation of primitive hematopoietic stem cells (HSC). HSC represent a very small subset of hematopoietic cells and provide self-renewal, possess differentiation capacity and allow a constant supply of the entire hematopoietic cell spectrum. Until recently, CD34 has been used as a convenient marker for HSC, since CD34(+) cells have been shown to possess colony-forming potential in short-term assays, maintain long-term colony-forming potential in in vitro cultures and allow the expression and differentiation of blood cells from different hematopoietic lineages in in vivo models. Clinical and experimental protocols have targeted CD34(+) cells enriched by a variety of selection models and have readily used these for transplantation, purging and gene therapies and targets for future organ replacement. Recent studies in murine and human models, however, have indicated that CD34(-) HSC exist as well, which possess engraftment potential and distinct HSC characteristics. These studies challenge the dogma that HSC are uniformly found in the CD34(+) subset, and question whether primitive HSC are CD34(+) or CD34(-). In this review, results on murine and human CD34(+) and CD34(-) HSC, differences between them and their possible interactions are examined.