Depending on the source of cells, the cell cycle status of hematopoietic stem and progenitor cells capable of repopulating the marrow of transplant recipients is controversial. In this study, using biochemical methods, the cell cycle status of mobilized CD34+ cells was analyzed. It was demonstrated in CD34+ cell extracts that there was high catalytic activity of G(1) cyclin-dependent kinases 4 and 6 (CDK4 and CDK6) but low activity of CDK2. This was in contrast to the resting reference cells that showed only minimal or no activity of these CDKs. Since at the G0-->G1-->S transition CDK4/6 and CDK2 sequentially phosphorylate the retinoblastoma protein (pRB), its phosphorylation status was analyzed. Previously, we showed that p110RB was unphosphorylated at serine (Ser)-608 in CD34+ cells, consistent with the ability to suppress cell growth. Here, it was established that this form of pRB was phosphorylated at Ser-780, Ser-795, and Ser-807/811 in CD34+ but not in resting reference cells. This result was therefore consistent with the presence of high CDK4/6 activities in CD34+ cells. Conversely, CDK2 activity was low and the pRB residues Ser-612 and threonine (Thr)-821, which are exclusively phosphorylated by CDK2 in conjunction with either cyclin E or A, were unphosphorylated in >90% of CD34+ cells. We therefore show for the first time the exact position of mobilized CD34+ cells within the cell cycle; that is, they do not reside in G0 but in early G1 phase and did not cross the restriction point into late G1 phase.