We have studied the function of the Hep III fibronectin domain in the cytoskeletal response initiated by alpha5beta1 integrin-mediated adhesion. Melanoma cells formed stress fibers and focal adhesions on the RGD-containing FNIII7-10 fragment. Coimmobilization of FNIII4-5, a fragment spanning Hep III and containing the alpha4beta1 ligand H2 with FNIII7-10, or addition of soluble FNIII4-5 to cells preattached to FNIII7-10, inhibited stress fibers and induced cytoplasmic protrusions. This effect involved alpha4beta1 since: 1) mutations in H2 reverted the inhibition; 2) other alpha4beta1 ligands (CS-1, VCAM-1), an anti-alpha4 mAb, or alpha4 expression in HeLa cells inhibited stress fibers. This activity was apparently cryptic in fibronectin or large fibronectin fragments, but exposed upon proteolytic degradation. Indeed purified peptic fragments containing H2, inhibited stress fibers when mixed with FNIII7-10 or fibronectin. RhoA activation with LPA or transfection with V14RhoA reverted the inhibitory effect and induced stress fibers on FNIII7-10+FNIII4-5. Furthermore, addition of alpha4beta1 ligands to FNIII7-10, down-regulated RhoA and activated p190RhoGAP, which localized to cytoplasmic protrusions. alpha4beta1/ligand interaction induced cell migration, monitored by video microscopy and wound healing assays. These data indicate that alpha4beta1 provides an antagonistic signal to alpha5beta1 by interfering with the RhoA activation pathway and this leads to melanoma cell migration.