Fibronectin and fibrinogen occur under the migrating epidermal tongue during reepithelialization of an excisional wound, and fibronectin increases in conjunction with capillary and fibroblast ingrowth during wound healing. Although we have previously shown that fibronectin is produced by proliferating blood vessels, the source of fibronectin associated with reepithelialization and fibroblast ingrowth has not been determined. In this report we demonstrate that subepidermal fibronectin derives mostly from plasma early in reepithelialization of an cxcisional wound and comes from both plasma and in situ production late in reepithelialization. This finding was established by extirpating 3 mm of skin from the center of a well-healed rat xenograph on the flanks of immunosuppressed mice, harvesting the open wound sites at 2, 4, 7, and 10 days after injury, and staining the specimens with reciprocal species-specific anti-fibronec-tin antibodies conjugated with fluorescein. In the first 4 days after wounding, newly forming rat epidermis migrated mainly over mouse fibronectin. In contrast, by 7 days after excision, the rat epidermis transits over a matrix containing both mouse and rat fibronectin, or rat fibronectin alone, indicating that a major component of the fibronectin is produced in situ. Although the biologic significance of these observations has not been fully elucidated, fibronectin may be part of a provisional matrix that functions to support, if not actively participate in, cell recruitment to sites of inflammation or wound healing.