We have evaluated the immunophenotype, functional activity and clonogenic potential of CD34+ cells from peripheral blood (PB) of normal donors before and after 4 and 6 days of G-CSF administration. The percentage and absolute number of CD34+ cells significantly increased at days 4 and 6 of G-CSF administration, compared to the steady-state level (P< 0.0001). two-colour fluorescence analysis showed, at days 4 and 6, a lower proportion of cd34+/c-kit+ compared to the steady-state level (P< 0.0001), but a similar expression of cd13, cd33, cd38, hla-dr and thy-1 antigens on cd34+ cells. The expression of adhesion molecules on CD34+ cells revealed a significant reduction of CD11a (P= 0.009), CD18, CD49d and CD62L (P< 0.0001) at days 4 and 6, compared to the baseline level. three-colour staining showed a reduction of the more immature compartment (34+/DR−/13−) and an increase of the more differentiated compartment (34+/DR+/13+). Downregulation of VLA-4 during mobilisation was seen almost exclusively on more committed cells (34+/13+); downregulation of CD62L, on the contrary, was observed on both early progenitors (34+/13−) and more committed cells (34+/13+). The expression of 34+/VLA-4+ decreased on both c-kit+ and c-kit− cells, while the expression of 34+/62L+ decreased on the c-kit+ cells only. In vivo administration of G-CSF reduced the adherence capacity of CD34+ cells to normal BM stroma; in vitro incubation with SCF or IL-3 enhanced the expression of CD49d on CD34+ cells, while GM-CSF reduced the expression of CD62L. SCF was the only cytokine able to induce a significant increase of CD34+ cell adherence to preformed stroma. Pre-incubation with the blocking β2 integrin monoclonal antibody caused a reduction of CD34+ cell adherence. In conclusion, the decrease of CD49d expression on mobilized CD34+ cells correlates with a poor adhesion to BM stroma; CD34+ cells incubated in vitro with SCF showed, conversely, a higher expression of CD49d and a greater adherence capacity on normal preformed stroma.