Perfused human bone marrow (BM) mononuclear cell (MNC) cultures result in a greater long-term culture-initiating cell (LTC-IC) output than parallel CD34+lin- cell cultures, even when CD34+lin- cells are placed on irradiated preformed stroma (IPFS). This difference has been attributed to accessory cell effects that are potentiated by medium perfusion. The present study investigated the relative contributions of direct contact- and soluble-mediated mechanisms of accessory cells in this culture system. CD34+lin- cells within (i.e., in contact with) the MNC accessory cell mixture generated greater LTC-IC output than CD34+lin- cells in contact with IPFS. Incubation of CD34+lin- cells with MNC conditioned medium (CM) resulted in partial restoration of MNC accessory activity, while CM from IPFS had no activity on LTC-IC output. Interestingly, the level of LTC-IC output supported by MNC CM was equivalent to that supported by direct contact with IPFS. CD34+lin- cells were then cultured in Transwell inserts either alone, with IPFS (direct contact), or with IPFS below the insert. Direct contact with IPFS significantly increased the output of cells, CFU-GM, and LTC-IC from CD34+lin- cells. IPFS below the insert also resulted in significantly increased cell and CFU-GM output, but did not significantly affect LTC-IC output. Further experiments using CM from CD34+lin- cells and IPFS cultures showed that LTC-IC supportive activity was present only when direct contact was allowed between CD34+lin- cells and IPFS. ELISA and RT-PCR experiments showed that contact did not induce changes in the levels of several known growth factors, including GM-CSF, IL-1beta, IL-3, IL-6, IL-11, LIF, KL, FL, Tpo, TGF-beta, and MIP-1alpha. These results indicate that direct contact between CD34+lin- cells and IPFS induces soluble activity, which specifically increases LTC-IC output from CD34+lin- cell cultures, providing evidence for a novel direct contact-mediated two-way mechanism of communication between primitive hematopoietic cells and stroma.