Using electron microscopy, we had previously demonstrated a very close transmembrane relationship between actin microfilaments and fibronectin fibrils, termed the fibronexus. Since vinculin, a recently discovered intracellular protein, is localized at the membrane-insertion regions of actin fibers, we studied its possible relationship to fibronectin and the fibronexus. Using double-label immunofluorescence microscopy, we have observed that the distributions of vinculin and fibronectin are strikingly coincident in normal Nil 8 hamster fibroblasts arrested in the G1 phase of the cell cycle, and in HSV-transformed Nil hamster cells treated with purified fibronectin after culturing in 0.3% serum. Extensively spread Nil 8 cells have numerous vinculin-positive focal patches, which are localized either directly over or in tandem with fibronectin fibers at the ventral surface. However, fibronectin and vinculin do not exhibit this relationship in Nil 8 cells grown in 5% serum. These vinculin patches closely resemble the vinculin plaques that Geiger found to be dark under interference-reflection microscopy, suggesting that fibronectin is associated with substrate-adhesion plaques in arrested cells. Fibronectin treatment of the HSV-transformed Nil cells cultured in a low concentration of serum results in the formation of ventral microprocesses, exhibiting an extraordinary congruence of vinculin and fibronectin staining. In addition, these cells bind matrix-like arrangements of fibronectin on their dorsal surface at sites of cell-cell interaction that are vinculin-negative. These results imply that two distinct types of fibronexuses may exist: a ventral substrate-adhesive nexus consisting of fibronectin, vinculin and actin, and a dorsal association matrix fibers. Transmembrane vinculin-fibronectin associations are evidently sensitive to the growth state of the cell.