Two distinct subsets of dendritic cells are produced within the non-adherent cell population of the stroma-dependent long-term culture system. These are the small subset containing dendritic cell precursors and their progeny, large long-term culture-dendritic cells, which resemble immature CD11c(+) CD11b(+) MHCIIlo CD8alpha(-) dendritic cells. The replicative and developmental potential of cells produced in long-term culture were investigated as a model for production of dendritic cells from progenitors. Cell proliferation and apoptosis were examined by labelling with bromodeoxyuridine and Annexin-V, respectively. The developmental potential of cells was analysed following transfer on to stromal monolayers or into in vitro colony and transwell assays. Results demonstrate that small long-term culture-dendritic cells are stromal cell-dependent. In the absence of stroma, they become apoptotic and die. Furthermore, direct contact with stromal cells is necessary for the differentiation and proliferation of small precursor cells. The small cell subset contains no long-term self-renewing cells, but instead appears to contain cells committed to developing into large long-term culture dendritic cells. The large long-term culture dendritic cell subset also contains dividing cells. Survival of large long-term culture-dendritic cells is dependent on soluble stroma-derived factor(s) and not direct contact with the stromal layer. All data suggest that the long-term culture system supports dendritic cell development from a self-renewing progenitor population resident within the stroma that gives rise to committed dendritic cell precursors and immature dendritic cells.