Stem cell maintenance in continuous bone marrow culture has been shown to be associated with close range interaction between hematopoietic cells and marrow derived adherent cells (MDAC). The extent to which the culture system is dependent upon this type of interaction has not been ascertained. Explants of non-adherent mouse bone marrow cells were inoculated into culture flasks bearing established monolayers of syngeneic MDAC. Aliquots of the same suspensions were injected into diffusion chambers which were then placed in the culture vessels, along with the free suspensions. It was found that the loss of CFUS after incubation for one week was significantly greater from the diffusion chambers than from the surrounding suspensions. These differences were not observed in cultures incubated over adherent layers of xenogeneic fibroblasts. The results indicate that close range cell:cell interaction is required for stem cell maintenance in continuous bone marrow culture.