Tumor cell interactions with fibronectin (FN) are important for the development of secondary tumors inside the bone marrow stroma. We studied and compared the in situ distribution of FN in paraffin-embedded human bone marrow sections and investigated the in vitro regulation of FN assemblage by bone marrow stromal cells (BMSC). Finally, the role of FN in the interaction of BMSC with tumor cells was studied. Fine elongated FN-positive cell extensions, probably of stromal cell origin, were observed as well as a limited amount of extracellular FN deposits in connective tissues around capillaries and sinusoids. In vitro studies, using the confocal laser scanning microscope, showed that BMSC produced a high amount of FN with a characteristic extracellular matrix formation in an extensive network. FN matrix formation was predominantly detected at contact sites between cultured BMSC. In in vitro cultures with low cell concentrations and in vivo with a limited number of stromal cell contacts only limited matrix was found. From previous studies it is known that the alpha5 beta1 integrin is involved in the regulation of FN assembly. Here the role of the alpha5-subunit of this integrin was investigated. By using two different monoclonal antibodies (mAb) against the alpha5-subunit (2H6 and mAb16) the assembly of endogenous FN was completely blocked, indicating that these antibodies are directed against the active epitope. Another mAb (mAb11) against the alpha5-subunit did not affect the FN assemblage. Codistribution analysis of alpha5-subunits, alpha v-subunits, actin, and FN demonstrated that the alpha5 beta1 integrin is associated with FN and not with intracellular actin. Integrins alpha(v) beta1, alpha(v) beta3, and alpha(v) beta5, also ligands of FN, did not colocalize with FN. Codistribution of alpha v with the terminal ends of actin and not with FN indicates that alpha(v)-subunits are mainly directed to vitronectin rather than to FN. The dominant role of alpha5 beta1 in FN interaction is underlined by effective blocking of tumor cell adhesion with BMSC using anti-alpha5, anti-beta1, and anti-FN antibodies. These results emphasize the important role of alpha5 integrin subunit in FN matrix assembly in human BMSC and an exclusive role of alpha5 beta1 in the anchorage and regulation of FN-mediated adhesion processes in the bone marrow.