We have studied normal and transformed cultured human fibroblasts by ultrastructural immunocytochemistry with antibodies to fibronectin (the large external transformation-sensitive glycoprotein). Human fibroblasts at low density have very little extracellular material organized into a filamentous form. A diffuse membrane component of low-density fibroblasts reacts with fibronectin antibodies. As normal fibroblasts grow to confluence, an extensive extracellular filamentous matrix forms. The filaments are 15 to 20 nm in diameter, lack periodicity, and frequently occur in a meshwork. Antibody to fibronectin reacts with these filaments as well as with a diffusely distributed membrane component. Cultures of SV40-transformed human fibroblasts lack the extracellular filamentous matrix reacting with fibronectin antibodies; however, fibronectin is detected on plasma membranes. These studies indicate that a major difference between normal and transformed human fibroblasts is the failure of transformed fibroblasts to form a fibronectin-containing extracellular filamentous matrix.