The aim of this study was to design the methodology of expansion and differentiation of CD34+ cells to monocytes/macrophages. The present study shows that optimal medium for expansion of CD34+ cells was X-VIVO 10 supplemented with FBS, SCF, IL-3, FLT-3L, TPO and 3-4 days culture. For differentiation of CD34+ cells to monocytes/macrophages was IMDM supplemented with FBS, M-CSF, SCF, IL-3, FLT-3L and culture for 7-10 days. Using this protocol it was possible to obtain 1000 - fold increase in number of expanded cells, of which 40-60% were CD14+, i.e. 600 - fold increase in the number of CD14+ cells. The presence of two subpopulations of monocytes: CD14+CD16- and CD14++CD16+, which occurred at the ratio of about 2:1 was observed. They are clearly distinct from known main subpopulations of blood monocytes CD14+CD16+ and CD14++. Supplementation of cultures with VD3 increased the percentage of CD14+CD16- and decreased the proportion of CD14++CD16+. Monocytes/macrophages, mainly CD14++/16+ subpopulation secreted IL-12, but not TNF and IL-10, induced allo-MLR (mainly CD14++/16+ subpopulation), fagocytosed S.aureus (mainly CD14+/16- subpopulation), but did not produce intracellular O2-. Hence, this study shows that it is possible to generate from CD34+ haematopoietic progenitors two subpopulations of human monocytes. They are clearly distinct from those present in the peripheral blood im ; plicating the existence of novel monocyte subsets with distinct biological function that represent different stages in monocyte differentiation.