Apligraf is a bilayered tissue-engineered product consisting of a bovine collagen matrix with neonatal fibroblasts, overlaid by a stratified epithelium containing living keratinocytes. The United States Food and Drug Administration has approved its use for venous leg ulcers and neuropathic diabetic foot ulcers. Apligraf provides a dermal matrix and produces cytokines similar to the human skin. However, its mechanism of action and ultimate fate in host wounds are unclear. The aim of this study was to evaluate the persistence of Apligraf fibroblasts and keratinocytes in human acute partial-thickness wounds (split-thickness donor sites) treated with Apligraf. In an open-label, within-patient, three-centered, controlled pilot study, 10 patients were treated with Apligraf, Apligraf dermis only (without epidermis), and a polyurethane film for donor site wounds of the same size, depth, and anatomical location. Apligraf DNA persistence was the primary outcome measure. Basement membrane components, cosmetic outcome, time to wound healing, and safety parameters were secondary outcome measures. One week after the initial treatment, reverse transcription polymerase chain reaction analysis found that two Apligraf and two Apligraf dermis-only-treated sites had Apligraf DNA present. Four weeks posttreatment, only one Apligraf and one Apligraf dermis-only sites showed the presence of Apligraf DNA. There was no difference between the three treatment modalities in establishing basement membrane in donor site wounds. No differences in other secondary outcomes were found. Apligraf DNA persisted in a minority of patients at 4 weeks in acute partial-thickness wounds. Apligraf's success in speeding healing of acute wounds appears to be related to factors other than the persistence of donor DNA or effect on basement membrane restoration.