The von Hippel-Lindau (VHL) tumor suppressor gene regulates extracellular matrix deposition. In VHL negative renal cancer cells, VHL(-), the lack of fibronectin matrix assembly is thought to promote and maintain tumor angiogenesis allowing vessels to infiltrate tumors. Therefore, and considering the importance of this process in tumor growth, we aimed to study why VHL(-) renal cancer cells fail to form a proper extracellular matrix. Our results showed that VHL(-) cells were not defective in fibronectin production and that the fibronectin produced by these cells was equally functional in promoting cell adhesion and matrix assembly as that produced by VHL+ cells. We have previously reported that VHL(-) cells fail to form beta1 integrin fibrillar adhesions and have a diminished organization of actin stress fibers; therefore, we aimed to study if the small GTPase family is involved in this process. We found that activation of the RhoA GTPase was defective in VHL(-) cells, and this was possibly mediated by an increased activation of its inhibitor, p190RhoGAP. Additionally, the expression of constitutively active RhoA in VHL(-) cells resulted in formation of a fibronectin matrix. These results strongly suggest an important role for RhoA in some of the defects observed in renal cancer cells.