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Actin cytoskeletal polymerization is associated with a pro-proliferative, pro-survival state. We hypothesized that the actin polymerization of wound cells is increased in the presence of wound matrix attachment and is decreased after disruption of this attachment. Musculocutaneous flap and wound splinting models were used to investigate the effect of wound matrix attachment on the actin cytoskeleton. Disruption of wound matrix attachment was accomplished by incision of the wound matrix/dermis interface (wound matrix release) and/or desplinting. Polymerized actin was assayed with phalloidin labeling of wound specimens 24 hours after disruption of attachment and a method to quantify the content and organization of polymerized actin in granulation tissue was used. Disruption of wound matrix attachment decreased the content of polymerized actin, the actin staining intensity, and the actin fiber organization in the granulation tissue of both the flap and splint models. Disruption of wound matrix attachment decreased actin polymerization and fiber organization in the granulation tissue. Our data support the concept that the state of wound matrix attachment regulates the actin cytoskeleton of wound cells.