beta1-null GD25 fibroblasts adherent to vitronectin fail to bind the N-terminal 70-kDa matrix assembly domain of fibronectin or to assemble fibronectin (Sakai, T., Zhang, Q., Fässler, R., and Mosher, D. F. (1998) J. Cell Biol. 141, 527-538). We have made four observations that extend this finding. First, the presence of vitronectin on a substrate that otherwise can support fibronectin assembly has a dominant-negative effect on assembly. Second, the dominant-negative effect is lost when active beta1A is expressed. Third, beta1A containing the extracellular D130A inactivating mutation has a dominant-negative effect on fibronectin assembly. Fourth, beta1-null cells adherent to vitronectin are flat and lack filopodia, whereas beta1-null cells adherent to fibronectin or beta1A-expressing cells adherent to either vitronectin or fibronectin are contracted and exhibit numerous filopodia. These results reveal, therefore, that GD25 cells adherent to vitronectin can only assume a shape suitable for assembly of fibronectin when there is a countervailing signal from functional beta1-integrins.