There is limited understanding of CD34− hematopoietic cells and the linkage between CD34 antigen expression and cell proliferation. In this study, early CD34− CD38− LIN− (CD34−) cells were purified from mobilized adult peripheral blood and carefully analyzed in vitro for growth and modulation of CD34. Mobilized CD34+CD38− LIN− (CD34+) cells were used for comparison. Expression of CD34, CD38, and LIN antigens was determined, and proliferative responses were assessed with PKH tracking dye, expression of Ki67 antigen, and uptake of pyronin Y. Suspension cultures of adult CD34− cells generated CD34+ cells and progenitors for >8 weeks. Stromal cultures demonstrated the presence of long-term culture-initiating cells within the CD34− fraction. While CD34− cells were slower to initiate growth than the CD34+cells were, no significant difference in hematopoietic cell output was found. Upon cultivation of CD34− cells, CD34 antigen appeared within 48 hours but was restricted to those cells that had initiated growth. Surprisingly, CD34+ precursors lost CD34 expression in culture if they remained in G0 for more than 2 days. Those cells later regained expression of CD34 antigen upon initiation of growth. Comparison of cells that did or did not rapidly modulate CD34 antigen revealed no differences in long-term growth potential. In conclusion, in vitro expression of CD34 by CD34− and CD34+ populations is tightly linked to cellular proliferation. In this culture system, expression of CD34 antigen by LIN− cells constitutes an early hallmark of growth. Measurement of CD34 expression by LIN− cells in expansion culture underestimates the total content of hematopoietic cells.