Current tissue culture techniques enable human keratinocytes (HK) from a small section of skin to be grown into sheets of epithelium for treating extensive wounds. The additional use of dermal replacements coupled with cultured skin substitutes may improve handling properties and effect ultimate healing results. Adhesion of cultured HK grafts to wounds, and final success rates of HK grafting, are variable and frequently unsatisfactory. To evaluate adhesion molecule (integrin) expression by cultured grafts, as well as matrix molecule distribution, we performed immunohistologic analysis of integrin expression on HK cultured on plastic and a polyurethane membrane, as well as on two dermal substitutes. Multilayered HK sheet grafts were prepared by culturing cells in plastic tissue culture dishes, and HK were cultured to single-layer confluence on polyurethane membranes (Hydroderm; Wilshire Medical Inc., Dallas, Texas). Composite grafts were prepared by seeding proliferating HK on Dermagraft, composed of human neonatal fibroblasts cultured in polyglactin mesh (Advanced Tissue Sciences, La Jolla, Calif.) and on AlloDerm, an acellular human dermis (LifeCell Inc., The Woodlands, Texas). Immunohistologic staining was performed for the integrin subunits alpha 5, alpha 6, and alpha v. Staining for matrix molecules included fibronectin, laminin-1, and laminin-5. HK in cultured epithelial sheets showed integrin alpha 6 expression on basal cells, and only faint alpha 5 and alpha v staining. HK cultured to confluence on Hydroderm reacted with monoclonal antibodies specific for alpha 5, alpha 6, and alpha v. Through HK adhered well to Dermagraft, there was reduced adhesion of HK on AlloDerm that was not accelerated by addition of fibronectin. HK in composite grafts showed distinct reactivity according to the time in culture. HK on Dermagraft lost alpha 5 reactivity by day 17 and only weak alpha v reactivity was seen. Basal keratinocytes on AlloDerm, however, remained alpha 5 and alpha v positive. In both composite grafts, integrin alpha 6 expression was limited to basal keratinocytes.