The epidermal growth factor receptor (EGFR) is known to bind to the F-actin cytoskeleton in intact cells, and this interaction has been suggested to sequester the EGFR signal transduction system to specific loci within the cell. In this study, the interaction of the EGFR with actin is examined in a reconstituted cell free system. Soluble protein components of the cytosol from A431 cells are shown to dramatically enhance binding of the EGFR to F-actin in a saturable, concentration-dependent fashion. Most of the intracellular C-terminal portion of the EGFR is found to be required for this interaction. Binding of the EGFR to F-actin strongly deactivates the receptor by diminishing EGFR autophosphorylation activity and enhancing tyrosine phosphatase activity toward the EGFR. These results suggest that a ternary or larger protein signaling complex forms on an F-actin cellular scaffold, providing a spatially restricted signal modulation site that can be regulated by cytoskeletal remodeling.