Regeneration of the human anterior cruciate ligament after complete rupture offers several theoretical advantages over reconstruction, including maintenance of the complex insertion sites and fan-shape of the ligament and preservation of remaining proprioceptive fibers within the ligament substance. Well vascularized connective tissues, such as dermis, heal as a result of migration of fibroblasts into a provisional scaffold, the fibrin clot. Wound closure is subsequently facilitated by a contractile cell phenotype. This study was designed to determine if fibroblasts intrinsic to the human anterior cruciate ligament were capable of migrating from their native extracellular matrix onto an adjacent provisional scaffold in vitro. Another objective was to determine whether any of the cells that successfully migrated into the scaffold expressed the contractile actin isoform, alpha-smooth muscle actin, associated with wound contraction in other tissues. The results demonstrated that the cells intrinsic to the human anterior cruciate ligament were able to migrate into a collagen-glycosaminoglycan scaffold, bridging a gap between transected fascicles in vitro. As a result of this cell migration and proliferation, areas in the scaffold contained cell number densities similar to those seen in the human anterior cruciate ligament in vivo. No extracellular matrix or tissue formation was seen in the gap between directly apposed transected ends of the anterior cruciate ligament explants cultured without an interposed collagen-glycosaminoglycan scaffold. The fascicle-collagen-glycosaminoglycan-fascicle constructs and the fascicle-fascicle explants displayed minimal adherence after 6 weeks in culture. Any disruption in the contact area between explant and scaffold, even as small a gap as 50 microm, prevented cell migration from the explant to the collagen-glycosaminoglycan scaffold at the area of loss of contact. All cells that migrated into the scaffold at early time periods expressed the alpha-smooth muscle actin isoform. These results demonstrate that cells that migrate into and proliferate within the collagen-glycosaminoglycan matrix have contractile potential as reflected in their expression of the alpha-smooth muscle actin isoform. The role of these contractile cells in the healing process warrants further investigation. Moreover, this study demonstrates the potential of cells intrinsic to the human anterior cruciate ligament to migrate into collagen-glycosaminoglycan scaffolds that may ultimately be investigated as implants to facilitate ligament healing and regeneration.